Luciferase-based assays

ABSTRACT

A method and kit is provided for enhancing the tolerance of an assay reagent to compounds in an assay sample, the assay reagent including a luciferase enzyme. The method includes contacting the luciferase with a tolerance enhancement agent in an amount sufficient to substantially protect luciferase enzyme activity from interference of the compound and minize interference by at least about 10% relative to an assay not having tolerance enhancement agent.

CROSS-REFERENCE

This application claims the benefit of priority from U.S. provisionalapplication No. 60/436,173, filed Dec. 23, 2002; No. 60/444,264, filedJan. 31, 2003; and No. 60/447,334, filed Feb. 13, 2003, all which areincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to bioluminescence. Moreparticularly, the invention concerns methods, compositions, and kits forimproving the accuracy of luciferase-based assays for high throughputscreening of compound libraries by reducing the number of “false hits”.The invention is particularly well suited for assays and test kits whichuse bioluminescence for quantitating products or occurrences of certainbiospecific reactions in cellular and cell-free systems.

BACKGROUND OF THE INVENTION

Advances in the biological, biomedical and pharmaceutical sciences haveaccelerated the pace of research and diagnostics to a level unparalleledto the past. With sequences of whole genome becoming available quicklyand successively, the assembly of large libraries of small molecules,the ability to move pharmaceutical development, clinical diagnostictests and basic research from a reductionist to a whole system approachquickly all demand assays that facilitate high throughput analyses.Molecules no longer need to be singly analyzed for their effects on alone process. Instead, the effects of many molecules on severalbiological systems can be studied simultaneously if appropriate, fast,reliable, and accurate assays are available.

Efficient, reliable and accurate assays for determining the occurrenceof a certain biospecific events, e.g., enzyme inhibition in a cell-freeenvironment or for assessing cell viability can be used to rapidlydiscover potential new pharmaceutical agents and to determine thecytotoxic effect or cell proliferation effect of such agents on cells.For instance, cancer pharmaceutical research often endeavors to identifycompounds that selectively kill rapidly dividing cells, a primarycharacteristic of cancer cells. High throughput screens of compoundlibraries, coupled with efficient cell viability assays, can swiftlyidentify such compounds as potential cancer drugs. The efficacy of acandidate compound on cell viability can be assayed by detecting ATPsince ATP production is realized only in metabolically active livecells; residual ATP is rapidly degraded upon necrotic cell death. SeeU.S. patent application Ser. No. 09/813,279, filed Mar. 19, 2001,entitled “Improved Method for detection of ATP” (assignee: Promega)which is incorporated by reference in its entirety. In another example,the identification of potential drug compounds that should move forwardin the drug development process can be made by determining the effectsof these compounds on cytochrome P-450 enzyme activity. See U.S. S. No.application Ser. No. 10/665,314, filed Sep. 19, 2003, entitled“Luminescence-based methods and probes for measuring cytochrome P-450activity” (assignee: Promega) which is incorporated by reference in itsentirety. In a final example, proteases represent a large and importantgroup of enzymes involved in diverse physiological processes includingblood coagulation, inflammation, reproduction, fibrinolysis, and theimmune response. The identification of protease inhibitors may be usefulfor the investigation, treatment or management of disease states causedby or characterized by the alteration in the activity of specificproteases. See U.S. Provisional application No. 60/353,158, filed Feb.1, 2002 entitled “Bioluminescent Protease Assay” (assignee: Promega),which is incorporated by reference in its entirety. Assay systems likethese not only facilitate the evaluation of a substance on cellviability or proliferation in a cellular environment or on theoccurrence of a biospecific reaction, but also permit high throughputscreens that can rapidly test thousands of compounds, streamlining newdrug discovery.

The use of reporter molecules or labels to qualitatively orquantitatively monitor molecular events is well established in assaysused for medical diagnosis, for the detection of toxins and othersubstances in industrial environments and for basic and applied researchin biology, biomedicine and biochemistry. Reporter molecules or labelsin such assay systems have included radioactive isotopes, fluorescentagents, enzymes, including light-generating enzymes such as luciferase.Desirable characteristics of any reporter molecule systems include safe,quick and reliable application and detection. Luminescent systems areamong the most desirable since they are exceptionally safe andsensitive.

Light-emitting systems have been known and isolated from manyluminescent organisms, including certain bacteria, protozoa,coelenterates, mollusks, fish, millipedes, flies, fungi, worms,crustaceans, and beetles. Those enzymes isolated from beetles,particularly the fireflies of the genera Photinus, Photuris and Luciolaand click beetles of genus Pyrophorus have found widespread use inreporter systems. In many of these organisms, enzymatically catalyzedoxidoreductions take place in which the free energy change is utilizedto excite a molecule to a high-energy state. When the excited moleculespontaneously returns to the ground state, visible light is emitted.This emitted light is called “bioluminescence” or “luminescence”.Luminescent luciferase-based assays have been developed to monitor ormeasure kinase activity, P450 activity, and protease activity. See, forinstance, U.S. application Ser. No. 10/665,314, filed Sep. 19, 2003(P450 activity); U.S. patent application Ser. No. 09/813,279, filed Mar.19, 2001 (kinase activity); and U.S. provisional application no.60,353,158, filed Feb. 1, 2002 (protease activity), commonly owned byPromega Corporation.

Genetic reporter systems are widely used to study eukaryotic geneexpression and cellular physiology. Applications include the study ofreceptor activity, transcription factors, intracellular signaling, mRNAprocessing and protein folding. Currently, luciferase genes from a widevariety of vastly different species, particularly the luciferase genesof Photinus pyralis (the common firefly of North America), Pyrophorusplagiophthalamus (the Jamaican click beetle), Renilla reniformis (thesea pansy), and several bacteria (e.g., Xenorhabdus luminescens andVibrio spp), are extremely popular luminescence reporter genes.Reference is made to Bronstein, et al. (1994) Anal. Biochem., Vol. 219,pp. 73-82, for a review of luminescence reporter gene assays. Fireflyluciferase is also a popular reporter for ATP concentrations, and inthat role is widely used to detect biomass. Various other reporterapplications of luciferases have been described in the scientificliterature. Luminescence may be produced by other enzymes when mixedwith certain synthetic substrates; such as alkaline phosphatase mixedwith adamantyl dioxetanes, or horseradish peroxidase mixed with luminol.

Luciferase genes are widely used as genetic reporters due to thenon-radioactive nature, sensitivity, and extreme linear range ofluminescence assays. For instance, as few as 10⁻²⁰ moles of the fireflyluciferase can be detected. Consequently, luciferase assays of geneactivity are used in virtually every experimental biological system,including both prokaryotic and eukaryotic cell cultures, transgenicplants and animals, and cell-free expression systems. Similarly,luciferase assays of ATP are highly sensitive, enabling detection tobelow 10⁻¹⁶ moles of ATP.

Luciferases generate light via the oxidation of enzyme-specificsubstrates, called luciferins. For firefly luciferase and all otherbeetle luciferases, this is done in the presence of magnesium ions,oxygen, and ATP. For anthozoan luciferases, including Renillaluciferase, only oxygen is required along with the luciferin. Generally,in luminescence assays of genetic activity, reaction substrates andother luminescence-activating reagents are introduced into a biologicalsystem suspected of expressing a reporter enzyme. Resultantluminescence, if any, is then measured using a luminometer or anysuitable radiant energy-measuring device. The assay is very rapid andsensitive, and provides gene expression data quickly and easily, withoutthe need for radioactive reagents. Reporter assays other than forgenetic activity are performed analogously.

The conventional assay of genetic activity using firefly luciferase hasbeen further improved by including coenzyme A (CoA) in the assay reagentto yield greater enzyme turnover and thus greater luminescenceintensity. (Promega Luciferase Assay Reagent, Cat. No. E1500, PromegaCorporation, Madison, Wis.; see U.S. Pat. No. 5,283,179, issued Feb. 1,1994.) Using this reagent, luciferase activity can be readily measuredin luminometers or scintillation counters. The luciferase reaction,modified by the addition of CoA to produce persistent light emission,provides an extremely sensitive and rapid assay for quantifyingluciferase expression in genetically altered cells or tissues.

Dual reporters are commonly used to improve experimental accuracy. Theterm “dual reporter” refers to the simultaneous expression andmeasurement of two individual reporter enzymes within a single system.In genetic reporting, examples that currently benefit from dual-reporterassays include individual cells or cell populations (such as cellsdispersed in culture, segregated tissues, or whole animals) geneticallymanipulated to simultaneously express two different reporter genes. Mostfrequently, the activity of one gene reports the impact of the specificexperimental conditions, while the activity of the second reporter geneprovides an internal control by which all sets of experimental valuescan be normalized. Normalizing the activity of the experimental reporterto the activity of the internal control minimizes experimentalvariability caused by differences in cell viability or transfectionefficiency. Other sources of variability, such as differences inpipetting volumes, cell lysis efficiency and assay efficiency, can beeffectively eliminated. Thus, dual reporter assays often allow morereliable interpretation of the experimental data by reducing extraneousinfluences.

In genetic reporting, examples that currently benefit from dual-reporterassays include individual cells or cell populations (such as cellsdispersed in culture, segregated tissues, or whole animals) geneticallymanipulated to simultaneously express two different reporter genes. Mostfrequently, the activity of one gene reports the impact of the specificexperimental conditions, while the activity of the second reporter geneprovides an internal control by which all sets of experimental valuescan be normalized.

Cell-free reconstituted systems that may benefit from dual-enzymereporter technology are cellular lysates derived for the simultaneoustranslation, or coupled transcription and translation, of independentgenetic materials encoding experimental and control reporter enzymes.Immuno-assays may, likewise, be designed for dual-reporting of bothexperimental and control values from within a single sample.

Currently, genes encoding firefly luciferase (luc), chloramphenicolacetyl transferase (CAT), beta-galactosidase (lacZ), beta-glucuronidase(GUS) and various phosphatases such as secreted alkaline phosphatase(SEAP) and uteroferrin (Uf; an acid phosphatase) have been combined andused as co-reporters of genetic activity. The following referencesprovide representative examples of these various reporter genes used incombined form for the purpose of dual-reporting of genetic activity: lucand GUS: Leckie, F., et al., 1994; luc and CAT, and luc and lacZ: Jain,V. K. and Magrath, I. T., 1992; CAT and lacZ: Flanagan, W. M. et al.,1991; SEAP and Uf: Kondepudi, et al., 1994. See also PromegaDual-Luciferase® Reporter Assay system as well as Promega pGL3Luciferase Reporter Vectors (available from Promega Corporation,Madison, Wis.) as well as U.S. Pat. Nos. 5,744,320 and 5,670,356(assignee: Promega Corporation), which are incorporated by reference intheir entirety.

When luciferase is combined with a sample for the purpose of detecting aproduct such as ATP or the occurrence of a biospecific event, e.g.,inhibition or activation of caspase or P-450 activity, either in anenzyme assay or single/dual reporter assay format, one or more of thecompounds in a chemical library used for high throughput drug screeningmay adversely interact with luciferase and thus interfere with theassay. For instance, in a caspase assay compounds that only inhibitcaspase will result in decreased luminescence and would not be easilydistinguishable from compounds that only inhibit luciferase activitywhich also decreases luminescence. There is a need for luciferase-basedassays with improved tolerance for compound interference, especiallywhen employed in high throughput screening procedures.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions and kits forimproved luciferase-based assays with enhanced tolerance forinterference by one or more compounds, particularly in high throughputscreening of compound libraries. The inventive compositions comprise aluciferase and a tolerance enhancing agent that substantially protectsluciferase activity from interference by one or more compounds thatinhibit or interact with luciferase during screening assays. An optionalATPase inhibitor may be used, particularly if the sample includes celllysates or enzyme mixtures with ATPase activity. Preferably, thetolerance enhancing agent is a detergent. The improved composition isuseful in any assay that employs luciferase as a component of an assayreagent for determining the effect of one or more compounds on purifiedenzymes, enzyme mixtures, cell lysates or extracts, and/or tissuehomogenates in a sample. For instance, the improved compositions may beused in methods for determining the effect of one or more compounds onkinase enzyme activity, protease activity, P-450 enzyme activity, andATP utilizing or generating enzyme activity contained in a sample.

The invention provides methods for enhancing the tolerance of aluciferase enzyme activity to compound interference in a luciferasebased assay. In one embodiment, the methods comprise contacting aluciferase enzyme with a tolerance enhancement agent in an amountsufficient to substantially protect luciferase activity frominterference of the compound.

The invention also provides methods of determining the effect of acompound on a non-luminogenic enzyme activity comprising: (a) providinga compound and a luminogenic molecule, wherein the luminogenic moleculeis a substrate of the non-luminogenic enzyme and a pro-substrate of aluciferase enzyme; (b) contacting the compound and the non-luminogenicenzyme so as to produce a first reaction mixture; (c) contacting thefirst reaction mixture with a reagent composition comprising luciferase,the luminogenic molecule, and a tolerance enhancement agent so as toproduce a second reaction mixture, wherein said tolerance enhancementagent being present in an amount effective to at least substantiallyprotect the activity of the luciferase from interference from thecompound; (d) detecting luminescence in the second reaction mixture; and(e) determining the effect of the compound on the non-luminogenic enzymeactivity, if any, result from the interaction of the compound with theenzyme by measuring and comparing the luminescence of the secondreaction mixture with a control reaction mixture. In one embodiment ofthe invention, the steps are conducted sequentially. In anotherembodiment of the invention, steps (b) and (c) are conductedsimultaneously.

In addition, the invention provides methods of determining the effect ofa compound on a non-luminogenic enzyme activity comprising: (a)providing a compound and a luminogenic molecule, wherein the luminogenicmolecule is a substrate of the non-luminogenic enzyme and apro-substrate of a luciferase enzyme; (b) contacting the compound, andthe non-luminogenic enzyme so as to produce a first reaction mixture;(c) contacting the first reaction mixture with the luminogenic moleculeso as to produce a second reaction mixture; (d) contacting the secondreaction mixture with a reagent composition comprising luciferase and atolerance enhancement agent so as to produce a third reaction mixture,wherein said tolerance enhancement agent being present in an amounteffective to at least substantially protect the activity of theluciferase from interference from the compound; (e) detectingluminescence in the third reaction mixture; and (f) determining theeffect of the compound on the non-luminogenic enzyme activity, if any,result from the interaction of the compound with the enzyme by measuringand comparing the luminescence of the third reaction mixture with acontrol reaction mixture. In one embodiment of the invention, the stepsare conducted sequentially. In another embodiment of the invention,steps (b) and (c), or steps (c) and (d), or steps (b) to (d) areconducted simultaneously.

In addition, the invention provides methods of determining the effect ofa compound on a non-luminogenic enzyme activity comprising: (a)providing a compound for testing and a reagent composition comprising aluminogenic molecule, wherein the luminogenic molecule is a substrate ofthe non-luminogenic enzyme and a pro-substrate of a luciferase enzyme;the non-luminogenic enzyme; luciferase; and a tolerance enhancementagent, wherein said tolerance enhancement agent being present in anamount effective to at least substantially protect the activity of theluciferase from interference from the compound; (b) detectingluminescence in the final reaction mixture; and (c) determining theeffect of the compound on the non-luminogenic enzyme activity, if any,result from the interaction of the compound with the enzyme by measuringand comparing the luminescence of the final reaction mixture with acontrol reaction mixture.

Furthermore, the invention provides methods of determining the effect ofa compound on a non-luminogenic enzyme activity comprising: (a)providing a luminogenic molecule and a compound for testing, theluminogenic molecule is a substrate for the non-luminogenic enzyme and apro-substrate for luciferase; (b) contacting the compound, theluminogenic molecule and a non-luminogenic enzyme so as to produce afirst reaction mixture; (c) contacting the first reaction mixture with areagent composition comprising luciferase, and a tolerance enhancementagent so as to produce a second reaction mixture, wherein said toleranceenhancement agent being present in an amount effective to at leastsubstantially protect the activity of the luciferase from interferencefrom the compound; (d) detecting luminescence in the second reactionmixture; and (e) determining the effect, if any, of the compound onnon-luminogenic enzyme activity resulting from the interaction of thecompound with the enzyme by measuring and comparing the luminescence ofthe second reaction mixture with a control reaction mixture. In oneembodiment of the invention, the steps are conducted sequentially. Inanother embodiment of the invention, steps (b) and (c) are conductedsimultaneously.

The invention also provides methods of determining the effect of acompound on a non-luminogenic enzyme activity comprising: (a) providinga compound for testing, a substrate for the non-luminogenic enzyme, anon-luminogenic enzyme, and ATP or ADP; (b) contacting the compound, thesubstrate, ATP or ADP, and a non-luminogenic enzyme so as to produce afirst reaction mixture; (c) contacting the first reaction mixture with areagent composition comprising luciferase, luciferin, and a toleranceenhancement agent so as to produce a second reaction mixture, whereinsaid tolerance enhancement agent being present in an amount effective toat least substantially protect the activity of the luciferase frominterference from the compound; (d) detecting luminescence in the secondreaction mixture; and (e) determining the effect, if any, of thecompound on non-luminogenic enzyme activity resulting from theinteraction of the compound with the enzyme by measuring and comparingthe luminescence of the second reaction mixture with a control reactionmixture. In one embodiment of the invention, the steps are conductedsequentially. In another embodiment of the invention, steps (b) and (c)are conducted simultaneously.

The invention further provides methods of determining the effect of acompound on ATP generating enzyme activity in a sample comprising: (a)providing ADP and a compound for testing; (b) contacting the compound,ADP and a sample so as to produce a first reaction mixture; (c)contacting the first reaction mixture with a reagent compositioncomprising luciferase, luciferin, and a tolerance enhancing agent so asto produce a second reaction mixture, wherein said tolerance enhancementagent being present in an amount effective to at least substantiallyprotect the activity of the luciferase from interference from thecompound; (d) detecting luminescence in the second reaction mixture; and(e) determining the effect, if any, of the compound on ATP generatingenzyme activity resulting from the interaction of the compound with theATP generating enzyme by measuring and comparing the luminescence of thesecond reaction mixture with a control reaction mixture. In oneembodiment of the invention, the steps are conducted sequentially. Inanother embodiment of the invention, steps (b) and (c) are conductedsimultaneously.

The invention further provides methods of determining the effect of acompound on ATP generating enzyme activity in a sample comprising: (a)providing a compound for testing, and contacting the compound with areagent composition comprising ADP, luciferase, luciferin, and atolerance enhancing agent, wherein said tolerance enhancement agentbeing present in an amount effective to at least substantially protectthe activity of the luciferase from interference from the compound; (b)detecting luminescence in the second reaction mixture; and (c)determining the effect, if any, of the compound on ATP generating enzymeactivity resulting from the interaction of the compound with the ATPgenerating enzyme by measuring and comparing the luminescence of thesecond reaction mixture with a control reaction mixture.

In a particular embodiment, a tolerance enhancement agent of theinvention can comprise a detergent, for example, a cationic, anionic,non-ionic, or zwitterionic detergent. In another embodiment, a toleranceenhancement agent can comprise a non-detergent, for example,polyethylene glycol, polyvinyl pyridine, crown ether, and cyclodextrin.In yet another embodiment, the detergent can comprise Tergitol®, Brij35®, Brij 58®, Triton X-100®, Triton X-305®, Triton N101®, CHAPS®,Chapso®, Bigchap®, Thesit®, Pluronic L64®, Rhodasurf 870®, Chemal LA-9®,Sulfonyl 465®, deoxycholate, CTAB, Pierce C08®, or Pierce C10®detergent.

In another embodiment, the methods of the invention further comprisecorrelating luminescence with enzyme concentration or activity.

In one embodiment, the enzyme activity is contained in a sample. In oneaspect, a sample can be a purified, partially purified or crude enzymeor mixtures of enzymes, a cell lysate, a tissue homogenate, or asubcellular fraction.

In another embodiment, a non-luminogenic enzyme in a method of theinvention can be a protease. In one aspect, the protease can be, forexample, trypsin, trypsinase, or caspase. In another aspect, the caspasecan comprise caspase-3, caspase-7, caspase-8 or caspase-9. In anotheraspect, the luminogenic substrate can comprise any masked substrate thatis a substrate of the protease and a prosubstrate of the luciferase suchas an amino-modified aminoluciferin or a carboxylprotected derivativethereof.

In still another embodiment, the non-luminogenic enzyme can be acytochrome P-450 enzyme, and the luminogenic substrate can be aD-luciferin derivative, and the tolerance enhancement agent can beTergitol® detergent, for example, Tergitol NP-9® detergent.

In yet another embodiment, the non-luminogenic enzyme can be a kinaseand the detergent can be Tergitol®, Thesit®, or CHAPS® detergent.

In another embodiment, the compound and enzyme in a method of theinvention can be contacted for a first predetermined time period priorto contact with the substrate and ATP or ADP. In one aspect, thesubstrate and ATP or ADP are added sequentially or simultaneously.

In yet another embodiment, an ATP generating enzyme can be a kinase orphosphatase and the tolerance enhancement agent comprises Tergitol®,Thesit®, or CHAPS®detergent.

The invention further provides methods of determining the effect of acompound on a kinase enzyme activity in an assay sample not containingliving cells, comprising: (a) providing a compound for testing, a kinasesubstrate, a kinase enzyme, and ATP or ADP; (b) contacting the compound,the substrate, ATP or ADP, and kinase enzyme so as to produce a firstreaction mixture; (c) contacting the first reaction mixture with areagent composition comprising luciferase, luciferin, and a toleranceenhancement agent so as to produce a second reaction mixture, whereinsaid tolerance enhancement agent being present in an amount effective tosubstantially protect the activity of the luciferase from interferencefrom the compound; (d) detecting luminescence in the second reactionmixture; and (e) determining the effect of the compound, if any, onkinase enzyme activity by comparing the detected luminescence to theluminescence of a similar reaction mixture not containing the compound,or containing the compound at a different concentration.

In a particular embodiment, the kinase enzyme is a protein kinase.

In one embodiment, the tolerance enhancement agent comprises aTergitol®, Thesit®, or Chaps® detergent.

In another embodiment, the compound and kinase enzyme are contacted fora first predetermined time period prior to contact with the substrateand ATP or ADP.

In another embodiment, the substrate and ATP or ADP are addedsequentially or simultaneously.

In yet another embodiment, the steps are carried out sequentially.

In yet another embodiment, steps(b) and (c) are carried outsimultaneously.

The invention also provides kits comprising, for example: (a) atolerance enhancement agent for substantially protecting luciferaseactivity from interference from a test compound; (b) optional luciferaseenzyme; (c) optional buffer reagents; and (d) directions for using thekit. In one embodiment, a kit of the invention further comprises ATP andmagnesium ions. In another embodiment, a kit of the invention furthercomprises luciferin, a cell lysing agent, and/or an ATP extractingagent.

These and other embodiments of the invention will become apparent inlight of the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the relief of inhibition on luciferase by detergentsin a standard luciferase-based reaction in the presence of potentialluciferase inhibitors tyrphostin AG494 or emodin. Part (a) compares RLUof control (no detergent) against reaction mixtures that contain 0.1 or1% of Tomah, Tergitol, Thesit, CHAPS, or Triton X-100 as a function ofrelative light unit values. Part (b) is an analysis that compares therelative % of control against reaction mixtures that contain 0.1 or 1%of Tomah, Tergitol, Thesit, CHAPS, or Triton X-100 FIG. 2 illustratesthe relief of inhibition on luciferase by a detergent, Tergitol, duringscreening of a known drug library using a standard luciferase-basedreaction. Part (a) compares the RLU of control (no detergent) againstreaction mixtures. Five drug compounds of the library where found toinhibit luciferase enzyme in the absence of Tergitol. Part (b) is arepeat of part (a) and compares the RLU in the presence of 0.1%Tergitol. Part (c) is a repeat of part (a) and compares the RLU in thepresence of 1% Tergitol. Part (d) is a bar graph that shows the dramaticeffect of Tergitol against four of the five drug inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Unless defined otherwise, all technical and scientific terms have thesame meaning as is commonly understood by one of skill in the art towhich this invention belongs. All cited patents and publications areincorporated by reference in their entirety unless otherwise noted.

The nomenclature recommendations of Demerec et al, 1966, where these arerelevant to genetics, are adapted herein. To distinguish between genes(and related nucleic acids) and the proteins that they encode, theabbreviations for genes are indicated by italicized (or underlined) textwhile abbreviations for the proteins start with a capital letter and arenot italicized. Thus, luc or LUC refers to the luciferase nucleotidesequence that encodes luciferase polypeptide or Luc.

An “isolated” or “purified” luciferase is one that has been identifiedand separated and/or recovered from a component of its naturalenvironment.

As used herein, the term “luciferase” refers to one or more oxygenasesthat catalyze a light emitting reaction. Thus, luciferase refers to anenzyme or photoprotein that catalyzes a reaction that producesbioluminescence. Luciferases of the invention can be recombinant ornaturally occurring, or a variant or mutant thereof, such as a variantproduced by mutagenesis that has one or more properties, such as thermalstability, that differ from the naturally-occurring protein.Non-limiting examples of naturally occurring luciferases include,luciferases found among marine arthropods, firefly luciferase, clickbeetle luciferase, and railroad worm luciferase. A non-limiting exampleof a luciferase photoprotein is the aequorin photoprotein.

The term “sample” as used herein, is used in its broadest sense andincludes, without limitation, a pure, partially purified or crude enzymeor mixtures of enzymes, cell lysates, subcellular fractions, or tissuehomogenates.

The term “detection,” as used herein, refers to quantitatively orqualitatively determining the effect of a test compound on the sample.

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues in one sequence that are identical to, with, oragainst amino acid residues in a second sequence in the region ofoverlap when the two sequences are optimally aligned. To determinepercent amino acid identity, sequences are locally aligned and ifnecessary, gaps are introduced to achieve the maximum percent sequenceidentity; conservative substitutions are not counted when calculatingsequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Publiclyavailable computer software such as BLAST software (available from theNational Center for Biotechnology Information, Bethesda, Md.) may beused to align peptide sequences. Those skilled in the art can determineappropriate algorithms and parameters for measuring alignment, includingany algorithms and parameters needed to achieve optimal alignment of twoamino acid sequences.

When amino acid sequences are aligned, the percent amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain percent amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:

% amino acid sequence identity ═(X/Y)⁻100 where X is the number of aminoacid residues scored as identical matches in the optimal alignment of Aand B by the sequence alignment program or algorithm and Y is the totalnumber of amino acid positions aligned.

B. Method, Composition and Kits

In one embodiment of the invention, the present invention providescompositions with properties comprising a luciferase, a toleranceenhancing agent to improve tolerance for compound interference, and/orone or more optional ATPase inhibitors. The tolerance enhancing agentincludes one or more substances such as detergents or sequesteringagents that improves the tolerance of the luciferase assay againstinterference by one or more test compounds during a screening procedure.Some of the tolerance enhancing agents, e.g., detergents, may alsofunction an enzyme anti-degradation agent, or as an ATPase inhibitor. Inthe case where the tolerance enhancing agent does not act as an ATPaseinhibitor and ATPase inhibition is desired, especially when sampleshaving cell lysates are used, one or more ATPase inhibitors may beincluded. The invention further provides methods using these novelcompositions to detect products or occurrence of a biospecific event ina sample measured as a single-step or as a multi-step protocol.Preferably the luminescence resulting from the combination of acomposition of the invention with a sample has an extended duration,i.e., diminished by less than about 50% relative to the luminescencejust after the composition is combined with the sample. The process ofthe invention significantly reduces the time and effort ofluciferase-mediated detection of the effects of compounds on a sample ordetection of occurrence of a biospecific event.

In general, the methods comprise adding a composition (“reagentcomposition”) comprising a luciferase (such as exemplified by, but arenot limited to SEQ ID Nos 1-4 described below), a tolerance enhancingagent, and/or one or more optional ATPase inhibitors (if the toleranceenhancing agent does not have ATPase inhibitory activity) to a sampleand detecting luminescence, wherein the activity of the reagent hasenhanced tolerance to luciferase inhibitors (i.e. the reagentcomposition is capable of protecting the luminescent signal frominhibition by at least 10% over the reagent in the absence of thetolerance enhancing component). Even more preferably, the toleranceenhancing agent would protect the luminescent signal by at least 30%,50%, 60%, 70%, 80%, 90%, 99% or greater. The reagent composition may beadmixed before use by adding a solution comprising one or more optionalATPase inhibitors to a lyophilized luciferase.

As mentioned above, the invention may be practiced in an environmentthat generates luminescence with extended duration. Loss of stability isdefined as irreversible loss of activity. The reagent composition losesstability over time and the amount of activity lost varies depending onthe particular luciferase, tolerance enhancing agent, optional ATPaseinhibitor and, when present, enzyme stabilizing agent used. In someinstances, the tolerance enhancing agent may have enzyme stabilizingability and thus may be used in place of a separate enzyme stabilizingagent. Preferably the stability of the reagent composition isdemonstrable in the temperature range of about 20° C. to about 37° C.Although the methods of the invention may be used with a samplecontaining any amount of ATP, it is preferable to use a samplecontaining a non-saturated amount of ATP (i.e., a range whereluminescence is linearly proportional to the concentration of ATP) inassays designed to detect ATP levels. For assays where luciferin levelsare measured such as in a Cytochrome P-450 or protease assay thatemploys pro-luciferin derivatives, it is desirable to employ saturatingATP levels since luciferin, not ATP, levels are being measured.

The luminescence generated by a luciferase reaction is typicallydetected with a luminometer although other detection means maybe used.The presence of light greater than background level indicates thepresence of ATP in the sample. The background level of luminescence istypically measured in the same matrix in which the sample exists, but inthe absence of the sample. Suitable control reactions are readilydesigned by one of skill in the art. Preferred luciferases used in thecompositions and methods of the invention generate a stable signal,i.e., they yield enhanced duration of luminescence in a luciferasereaction defined as less than 50% loss of luminescence per hour relativeto the luminescence at the time the luciferase reaction was initiated.Preferred luciferases of the invention allow for multiple analyses of asample over time or analysis of many samples over time, one hour afterthe luciferase is combined with the reagent composition, more preferablytwo hours and most preferably four hours or more. Optionally, theluciferases used in the compositions and methods of the invention haveenhanced thermostability properties.

Quantifying the amount of emitted light also quantifies the amount ofATP in a sample, and thereby light can be used to quantify living cells.Quantitative ATP values are realized, for example, when the quantity oflight emitted from a test sample is compared to the quantity of lightemitted from a control sample or to a standard curve determined by usingknown amounts of ATP and the same luciferase, substrate, and reactionconditions (i.e. temperature, pH, etc.). It is understood thatquantification involves subtraction of background values. QualitativeATP values are realized when the luminescence emitted from one sample iscompared to the luminescence emitted from another sample without a needto know the absolute amount of ATP present in the samples, e.g., acomparison of samples in the presence or absence of a test compound.Many such experiments can readily be designed by one of ordinary skillin the art

Examples of ATPase inhibitors include detergents, preferably detergentswith charged groups such as cationic detergents [e g, DTAB(dodecyltrimethylammonium bromide), CTAB (cetyltmethylammonium) andBDDABr (benzyldimethyldodecylammonium bromide)], anionic detergents(e.g., SDS and deoxycholate), and zwitterionic detergents (e g,sulfobetaine 3-10)]. To facilitate the method, a substrate for theluciferase, such as luciferin, may be included in the reagentcomposition. Other embodiments of the reagent composition may furthercomprise a compound that prevents an increase in ATP levels in thesample over time. Compounds that prevent an increase in ATP levels inthe sample include NaF, vanadate and para-nitrophenylphosphate. Stillother embodiments of the reagent composition further comprise a bufferand magnesium. One of skill in the art knows that other cations, such asmanganese and calcium, may be suitable substitutes for magnesium.

The reaction composition may also comprise an optional enzymestabilizing agent. The enzyme stabilizing agent can be any compound thatstabilizes the luciferase from degradation. Suitable enzyme stabilizingagents include proteins (such as bovine serum albumin or gelatin) ordetergents (preferably non-ionic detergents, most preferably THESIT).

Further, the present invention is useful for determining the effect ofsmall molecules (including organic and inorganic molecules and syntheticand naturally occurring molecules) on cell free enzyme assays, which inturn allows the assessment of whether the small molecule may function asa pharmaceutical. The invention is directed to methods that determinethe effect of a small molecule or compound on a cell-free enzyme. One ofskill in the art may develop many other such assays for which theinvention is useful.

The invention also assembles the elements of the invention into kits.Such kits are designed to determine the effect of a compound or theoccurrence of a biological event within a sample, e.g. determining theeffects of compounds on enzymes. Kits can be multifunctional such thatmore than one purpose can be realized. In one embodiment, a kitcomprises lyophilized luciferase in one container, while anothercontainer contains reconstitution buffer with one or more toleranceenhancing agents and/or one or more ATPase inhibitors. The toleranceenhancement agents and/or ATPase inhibitors may be selected from thesubstances and detergents already disclosed, for example, DTAB, BDDABr,SDS, deoxycholate, or sulfobetaine 3-10, or a combination thereof.

The kit may also supply a luciferase substrate, such as luciferin,coelenterazine, or a functional derivatives thereof. The kit may alsosupply magnesium or other cations such as manganese or calcium. Tofacilitate the use of control experiments with known concentrations ofATP, such as in embodiments of the kits that are used to quantify ATP ina sample, a container that has ATP may also be supplied in such kits.The kit may also supply a compound that prevents an increase in theamount of ATP in the sample over time (e.g., NaF). The kit may alsosupply an ATP-ase inhibitor (e.g., TCA, DMSA, CTAB, ethanol, and thelike). The kit may also supply a buffer. The kit may also supply anenzyme stabilizing agent, e.g., BSA or gelatin or THESIT.

The kit may contain components which, when combined, generate a reagentcomposition that (i) maintains at least about 30% (preferably at leastabout 60%, even more preferably at least 70%, 80%, 90%, 95%, 99%)activity for at least about one hour (preferably at least two hours,more preferably four hours), as detected by luminescence when thereagent composition is combined with a sample, and relative to thereagent composition's activity just after it is assembled (i.e., 0 to 10minutes after the component comprising luciferase is combined with thecomponent comprising an tolerance enhancing agent and optional ATPaseinhibitor) and (ii) reduces at least about 25% or at least about 30%,(preferably at least about 40%, even more preferably at least about 50%,60%, 70%, 80%, 90%, 95%, 99% or any increment therein) of the ATPaseactivity that is endogenous to the sample relative to the sample'sATPase activity in the absence of the ATPase inhibitor.

The component comprising an ATPase inhibitor may comprise greater thanone ATPase inhibitor wherein they are present in the reagent compositionat a concentration such that their combined effect reduces at leastabout 25% or at least about 30%, (preferably at least about 40%, evenmore preferably at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or anyincrement therein) of the ATPase activity that is endogenous to thesample relative to the sample's ATPase activity in the absence of theATPase inhibitor and when allow for the reagent composition.

The kit preferably comprises a container comprising a buffered detergentsolution, said buffered detergent solution at a pH in the range of aboutpH 6.0 to about pH 9.0. In one embodiment of the invention, saidbuffered detergent solution comprising DTAB whose concentration in thereagent composition is in the range of about 0.05% to about 2% (w/v) andoptionally comprising NaF whose concentration in the reagent compositionis in the range of about 1 mM to about 20 mM and optionally comprisingTHESIT, a tolerance enhancing non-ionic detergent agent, whoseconcentration in the reagent composition is in the range of about 1% toabout 5%. The kit additionally comprises a separate container comprisinglyophilized luciferase, preferably a luciferase with the sequence of SEQID Nos: 1, 2. 3, or 4, most preferably SEQ ID Nos: 2 or 4. Preferablythe luciferase, when combined with the buffered detergent solutioncreating the reagent composition, is at a concentration of 1 μg/ml orgreater, more preferably at a concentration of 80 μg/ml or greater.Preferably, the container comprising lyophilized luciferase furthercomprises lyophilized luciferin. Optionally, the kit further comprisesinstructions for use of the kit for the purpose of measuring ATP.

In preferred embodiments, the present invention reduces to a single stepthe manipulations needed for determining enzyme, e.g., kinase, activityin a sample in the presence of one or more test compounds, prior toluminescence measurement. In the single-step ATP assay of the invention,all of the necessary components of the ATP-dependent enzyme (e.g.,luciferase), such as the enzyme, substrates, tolerance enhancersubstances and ATPase inhibitors are comprised within a reagentcomposition and are added to a sample at once. In some embodiments, acomponent of the reagent composition is an enzyme stabilizing agent.

In another embodiment of the invention, methods, compositions and kitsare provided for determining the effect of one or more compounds,preferably in a high-throughput screening format, on enzyme, e.g.,protease, activity by detecting and quantifying luciferin levels in asample. The method comprises adding to a sample a composition (“reagentcomposition”) comprising a luciferase enzyme, a tolerance enhancingagent to improve tolerance for compound interference, and a substratefor the protease that is a prosubstrate for the luciferase such as anamino-modified aminoluciferin or carboxylprotected derivative thereof,and detecting luminescence produced in the sample by the conversion of asubstrate-derived aminoluciferin into a luminescing compound byluciferase. These masked luciferase substrates are cleaved and unmaskedby the protease. For instance, the amino-modified aminoluciferin orderivative thereof has a covalent linkage of a protease substrate to theamino group of aminoluciferin and the protease is capable of cleavingits substrate at the covalent linkage or peptide bond, releasingaminoluciferin, a substrate for luciferase. U.S. patent application No.60/353,158, filed Feb. 1, 2002 entitled “Bioluminescence protease assay”(assignee: Promega Corp.), incorporated herein in its entirety,describes luciferase-based assays for determining protease activity anduseful protease substrates.

In one aspect of this invention, a sensitive luminescent method isprovided to determine protease activity, e.g., a caspase, trypsin ortryptase, in the presence of one or more compounds. For instance, theinvention provides a luminescent assay method to determine the activityof one or more caspases in the presence of one or more compounds. Themethod comprises contacting a sample suspected of having one or morecaspases with a mixture comprising beetle luciferase and anamino-modified beetle aminoluciferin or a carboxyl protected derivativethereof, and a tolerance enhancing agent wherein the amino group ofaminoluciferin or the derivative thereof is modified so as to covalentlylink a substrate for the caspase or the carboxyl protected derivativethereof via a peptide bond to aminoluciferin. If the sample comprises acaspase having a recognition site in the substrate, the substrate iscleaved at the peptide bond that links the substrate to aminoluciferin,yielding aminoluciferin, a substrate for the luciferase, in the mixture.Luminescence is then detected. The method further comprises correlatingluminescence with protease concentration or activity, i.e., increasedluminescence correlates with increased protease concentration oractivity. As defined herein, the term “functional equivalent” of areference substrate is a substrate having one or more amino acidsubstitutions relative to the sequence of the reference substrate, whichfunctionally equivalent substrate is recognized and cleaved by the sameprotease at a substantially similar efficiency as the referencesubstrate. Preferably, the increased protease assay sensitivity withmethods employing the luminescent substrates described in U.S. patentapplication no. 60/353,158, filed Feb. 1, 2002 is at least 2 times, morepreferably 3, 4, 5, 6, 7, 8, 9, or 10, or even greater, for instance, atleast 15, 20, 25, 30, 40, 50, 100, 200, 500, or 1000 times or more,greater than that of a comparable assay employing a conjugate comprisinga fluorophore covalently linked to at least one substrate molecule or afunctional equivalent thereof. Thus, the methods of the invention maydetect less than 5 uU, or less, e.g., less than 1 uU, 0.5 uU or 0.2 uUof caspase in a sample. As used herein, the limit of detection means 3standard deviations above background noise (“noise” is 1 standarddeviation of background and background is a control without caspase).

Using a substrate for caspase 3 and 7 that was linked to eitheraminoluciferin or rhodamine-110, it was found that the limit ofdetection for the aminoluciferin-based substrate was 0.2-0.5 uU ofpurified caspase while that for the rhodamine-110-based substrate was 10uU. It was also found that the limit of detection of caspase expressingcells with the aminoluciferin-based substrate was 15 cells at 1 hourwhile the limit of detection for the rhodamine-110-based substrate was150 cells at 1 hour. The methods of the invention may be employed with asample comprising purified, partially-purified, or crude preparations ofenzyme.

The invention also provides a luminescent assay method to detect aprotease activity where the protease that specifically cleaves asubstrate comprising aspartate. The method comprises contacting a samplehaving one or more aspartate-specific proteases and including one ormore compounds with a mixture comprising luciferase, a toleranceenhancing agent, and an amino-modified aminoluciferin or a carboxylprotected derivative thereof, wherein the amino group of aminoluciferinor the derivative thereof is modified so as to covalently link thesubstrate via a peptide bond to aminoluciferin or a carboxyl protectedderivative thereof. The protease having aspartate as a recognition sitewill cleave the substrate at the peptide bond that links the substratecomprising aspartate to aminoluciferin, yielding aminoluciferin, asubstrate for the luciferase in the mixture. Then luminescence isdetected in the sample. Preferred proteases that specifically cleave asubstrate comprising aspartate include but are not limited to caspases,e.g., any one of caspases 1-14. Preferred substrates compriseX₁-X₂-X₃-D, wherein X₁ is Y, D, L, V, I, A, W, or P; X₂ is V or E; andX₃ is any amino acid, for instance, a substrate comprising DEVD, WEHD,VDVAD, LEHD, VEID, VEVD, VEHD, IETD, AEVD, LEXD, VEXD, IFEHD, or PEHD.

The invention also provides a luminescent assay method to determinetrypsin or tryptase activity in the presence of one or more compounds.The method comprises contacting a sample having trypsin or tryptase andone or more test compounds with a mixture comprising luciferase and anamino-modified aminoluciferin or a carboxyl protected derivativethereof, and a tolerance enhancer agent, wherein the amino group ofaminoluciferin or the derivative thereof is modified so as to covalentlylink a substrate for trypsin or tryptase via a peptide bond toaminoluciferin or a carboxyl protected derivative thereof. Luminescenceis then detected. Preferably, the luminescent assay is more sensitivethan a corresponding assay with a conjugate comprising a fluorophorecovalently linked to at least one substrate molecule or a functionalequivalent thereof. For trypsin, arginine and lysine are functionallyequivalent substrates as trypsin cleaves the peptide bond after thoseresidues with substantially similar efficiencies. The increased assaysensitivity with methods employing the luminescent substrates of theinvention for trypsin or tryptase is at least 2 times, more preferably3, 4, 5, 6, 7, 8, 9, or 10, or even greater, for instance, at least 15,20, 25, 30, 40, 50 or 100 times or more, greater than that of an assayemploying a conjugate comprising a fluorophore covalently linked to atleast one substrate molecule or a functional equivalent thereof. Using asubstrate for trypsin, it was found that the limit of detection for alysyl-aminoluciferin substrate was 3.0 pg while that for thearginine₂-rhodamine-110-based substrate was 12-30 pg. Thus, a trypsinassay that employs an amino-modified aminoluciferin substrate is atleast 4 times more sensitive than a corresponding assay with a conjugatecomprising rhodamine-110 covalently linked to two functionallyequivalent trypsin substrates.

Further provided is a luminescent assay method to determine the activityof a protease that specifically cleaves a substrate comprising arginineor lysine in the presence of one or more test compounds. The methodcomprises contacting a sample having one or more proteases specific fora substrate comprising arginine or lysine and one or more test compoundswith a mixture comprising luciferase, a tolerance enhancing agent, andan amino-modified aminoluciferase or a carboxyl protected derivativethereof covalently linked via a peptide bond to a substrate comprisingarginine or lysine and a tolerance enhancer agent. Luminescence in thesample is then detected. Preferably, the assay is more sensitive than acorresponding assay with a conjugate comprising a fluorophore covalentlylinked to the substrate or a functional equivalent of the substrate. Astryptase is released from activated mast cells in association withinflammatory conditions including allergic reactions such asanaphylactic reactions and allergic rhinitis, and trypsin in stool maybe indicative of cystic fibrosis, the methods of the invention isparticularly useful for screening test compounds that may be useful foranti-inflammatory therapy.

Kits useful in the methods of the invention are also envisioned. Suchkits may comprise the amino-modified aminoluciferins or carboxylprotected derivatives of the invention, and instructions for their use,a luciferase, a tolerance enhancing agent and also optionally a bufferfor a luminescence reaction.

In another embodiment of the invention, a method is provided formeasuring P450 activity in the presence of one or more test compounds. Areaction mixture comprising one or more P-450 enzymes and one or moretest compounds is prepared and incubated for a predetermined timeperiod. Thereafter, the mixture is contacted with a luminogenic moleculeand incubated for a predetermined time period. The cytochrome P450metabolizes the luminogenic molecule into a substrate for thebioluminescent enzyme in a first reaction. The reaction mixture is thencontacted with luciferase and a tolerance enhancing agent. Thebioluminescent enzyme then acts on the substrate in a second lightemitting reaction. Cytochrome P-450 activity is indirectly determined bymeasuring the amount of luminescence that is generated from the assaymixture relative to a control mixture. Controls may involve replacementof P450 enzyme with water or the P450 buffer, replacement of recombinantP450 membrane preparation with a similar preparation that lacks P-450enzyme, elimination of NADPH, or heat denaturation of P-450 enzyme priorto addition of the luciferin substrate. Luminescence can be measuredafter a predetermined incubation time period or continuously from thetime the reaction is initiated. U.S. patent application Ser. No.10/665,314, filed Sep. 19, 2003, entitled “Luminescence-based methodsand probes for measuring cytochrome P-450 activity” (assignee: PromegaCorp.), incorporated herein in its entirety, and U.S. patent applicationSer. No. 10/665,314, filed Sep. 19, 2003, the disclosure which is herebyincorporated by reference in its entirety, describe luciferase-basedassays for determining P450 activity and useful luminogenic substrates.

P450 activity can be determined using luminogenic molecules that areP450 substrates or a P450 substrate/bioluminescent enzyme pro-substrate,such as, a beetle luciferin or a luciferin derivative. The term“luciferin derivative” as used herein refers to a type of luminogenicmolecule or compound having a substantial structure of D-luciferin andmaybe a substrate of one or more cytochrome P450 enzymes and apro-substrate of luciferase. In the presence of cytochrome P450, thecompound is metabolized into luciferin, a substrate of luciferase. Inthe absence of prior P450 metabolism, some of the compound(s) may bindto luciferase as evidenced by their capacity to inhibit a reaction withluciferin, however, they are not turned over as substrate inlight-generating reactions. Without being bound by any theory ofoperation, it is believed that these compounds are most likelycompetitive inhibitors of luciferase. Useful, but non-limiting,D-luciferin derivatives are disclosed in U.S. patent application Ser.No. 10/665,314, filed Sep. 19, 2003, the disclosure which is herebyincorporated by reference in its entirety.

In another embodiment of the invention, a method is provided formeasuring the activity of a cytochrome P450 enzyme. A luminogenicmolecule that is a P450 substrate and a bioluminescent enzymepro-substrate can be contacted with one or more cytochrome P450 enzymesand bioluminescent enzyme, either simultaneously or in a stepwisemanner, for a predetermined time. In the presence of P450, theluminogenic molecule is metabolized into a substrate for thebioluminescent enzyme in a first reaction. The bioluminescent enzymethen acts on the substrate in a second light emitting reaction.Cytochrome P450 activity can be determined by measuring the amount ofluminescence that is generated from reaction mixture relative to acontrol (e.g., no P450 enzyme). For the P450 reaction to occur, P450reductase, NADPH and Mg⁺² are generally present in the system.Similarly, the presence of ATP and Mg⁺² is generally necessary forfirefly luciferase activity but not for Renilla luciferase activity. Anysuitable concentration of luminogenic molecule may be employed in thereaction mixture. In practicing this invention, the concentration of theluminogenic molecule generally ranges between about 10 nM to 1 mM,preferably in the linear range of the substrate dose response by aparticular P450 isoform, most preferably at the Km for the particularsubstrate/P450 isoform reaction or at Vmax for that reaction.

P450 activity can also be determined using luminogenic molecules thatare natural coelenterazine and coelenterazine derivatives (collectivelyreferred to as coelenterazines). Coelenterazines are known to luminescewhen acted upon by a wide variety of bioluminescent proteins,specifically marine luciferases. Examples of marine luciferases includeRenilla luciferase, aequorin, Gaussia luciferase, Oplophorus luciferase,and Cypridina luciferase. Useful, but non-limiting, coelenterazines aredisclosed in U.S. patent application Ser. No. 10/053,482, filed Nov. 2,2001, the disclosure which is hereby incorporated by reference in itsentirety. Coelenterazines are available from Promega Corporation,Madison, Wis. and from Molecular Probes, Inc., Eugene, Oreg.Coelenterazines may also be synthesized as described for example inShimomura et al., Biochem. J. 261: 913-20, 1989; Inouye et al., Biochem.Biophys. Res. Comm. 233: 349-53, 1997; and Teranishi et al., Anal.Biochem. 249: 37-43, 1997.

The P450 acts on these coelenterazines in one of two ways. In onereaction pathway, the luminogenic molecules are P450 substrates andbioluminescent enzyme pro-substrates and do not exhibit thecharacteristic coelenterazine chemiluminescent (luminescence in theabsence of a bioluminescent enzyme, e.g. Renilla-type luciferase). P450metabolism of the luminogenic molecule in a first reaction generates thesubstrate for the Renilla luciferase. The Renilla luciferase then actson the substrate in a second light-emitting reaction. P450 activity isthen ascertained by measuring the luminescence of the reaction mixturerelative to a control reaction mixture. In the second reaction pathway,coelenterazine or coelenterazine derivatives exhibit chemiluminescenceand are substrates for Renilla-type luciferase. P450 metabolism of sucha luminogenic molecule results in the loss of chemiluminescence andactivity with Renilla-type luciferase. In both types of reactionpathways, P450 activity may be detected either directly by a change inchemiluminescence by the action of the P450 alone or indirectly by achange in bioluminescence from a Renilla-type luciferase. Useful, butnon-limiting, coelenterazines are disclosed in U.S. patent applicationSer. No. 10/053,482, filed Nov. 2, 2001, the disclosure which is herebyincorporated by reference in its entirety as well as in U.S. patentapplication Ser. No. 10/665,314, filed Sep. 19, 2003, the disclosurewhich is hereby incorporated by reference in its entirety.

In another aspect of this invention, a method for determining P-450activity in the presence of one or more test compounds is provided.According to this method, a reaction mixture comprising one or moreP-450 enzymes, one or more test compounds, and a luminogenic molecule isprepared and incubated for a predetermined time period. The reactionmixture is then incubated for a predetermined time period. The P-450enzyme metabolizes the luminogenic molecule and converts it into asubstrate for the bioluminescent enzyme. Thereafter, the second reactionmixture is contacted with a luciferase and a tolerance enhancing agentfor a predetermined time period. The luciferase enzyme acts on thesubstrate in a second light emitting reaction. Cytochrome P-450 activityis then indirectly determined by measuring the amount of luminescencethat is generated from the reaction mixture relative to a control (e.g.,no P-450 enzyme). The tolerance enhancing component permits moreaccurate quantitation of the Cytochrome P-450 activity if the testcompound interacts directly with the luciferase instead of or inaddition to the Cytochrome P-450.

In yet another embodiment of the invention, methods, compositions andkits are provided for determining the effect of one or more compounds,preferably in a high-throughput screening format, on an ATP generatingenzyme activity. The method comprises adding to a sample including ADP,an ATP generating enzyme, and a compound, a composition (“reagentcomposition”) comprising a luciferase enzyme, a tolerance enhancingagent to improve tolerance for compound interference, and optionally anATPase inhibitor, and detecting luminescence produced in the sample bythe conversion of a substrate (luciferin) into a luminescing compound byluciferase. Representative of ATP generating enzymes include, withoutlimitation, phosphoglycerate kinase or phosphopyruvate kinase. See U.S.Pat. No. 6,235,480, issued May 22, 2001 (assignee: Promega Corporation),which is incorporated by reference in its entirety, describing aluciferase-based assay for determining ATP generating enzyme activity.The reagent composition of the present invention comprises a toleranceenhancing agent to improve tolerance for compound interference and/orone or more optional ATPase inhibitors, preferably a detergent, and anon-endogenous ATP-dependent enzyme, wherein the composition is capableof maintaining at least about 30% enzymatic activity for at least aboutone hour, preferably at least about 2 hours, more preferably at leastabout 4 hours, compared to its activity just after (0 to 10 minutes) theenzyme is combined with the ATPase inhibitor, and wherein the one ormore ATPase inhibitors are present in the composition at a concentrationsufficient to collectively reduce ATPase activity endogenous to thesample by at least about 25%, more preferably at least about 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 99% or any increment therein relative tothe ATPase activity endogenous to the sample in the absence of theATPase inhibitor. In preferred embodiments of the invention, thenon-endogenous ATP-dependent enzymes are luciferases.

In a preferred embodiment, the components of the reagent composition ofthe invention can be supplied as two parts that are admixed shortlybefore use: (1) a part comprising luciferase and (2) a part comprising atolerance enhancing agent for improving luciferase tolerance againstinterference from one or more compounds and one or more optional ATPaseinhibitors. The tolerance enhancing agent may have ATPase inhibitionactivity and thus may be used in place of a separate ATPase inhibitor.The presence of an ATPase inhibitor is desirable with cell lysatesamples. The luciferase component may further comprise luciferin andpreferably is lyophilized. The luciferase component optionally comprisesexcipients for lyophilization, protein (luciferase) stabilizer,magnesium (or alternative cation), and a magnesium chelator (oralternative cation chelator). The ATPase inhibitor component may furthercomprise a buffer, divalent cation metal chelators, magnesium (oralternative cation), a defoaming agent, anti-ATP-generating enzymeagents (e.g., NaF), an enzyme stabilizer (e.g., THESIT) and an agent forwhile in the presence of an ATPase inhibitor (and, optionally, kinaseinhibitors) that stabilizes the amount of ATP present in the sampleresults in a reliable and efficient method for detecting and quantifyingcellular ATP for extended periods of time.

C. Luciferases

Luciferase enzymes, whose catalytic products include light, offer highsensitivity, a detectable product and enable easy measurement of ATP orother molecule such as luciferin or luciferin derivative. However, anyluminescence-producing enzyme may be used in the methods andcompositions of the present invention.

At their most basic level, luciferases are defined by their ability toproduce luminescence. More specifically, a luciferase is an enzyme thatcatalyzes the oxidation of a substrate, luciferin, thereby producingoxyluciferin and photons.

To date, five classes of luciferases have been identified (Jones et al.,1999; Thomson et al., 1997). Of these, beetle luciferases, such as thatof the common firefly (family Lampyridae), form a distinct class withunique evolutionary origins (McElroy et al., 1969; White et al., 1969;White et al., 1975). Beetle luciferases are often referred to as fireflyluciferases in the literature; however, firefly luciferases are actuallya subgroup of the beetle luciferase class. Beetle luciferases may bepurified from the lanterns of the beetles themselves or from proteinexpression systems well known in the art (Baldwin and Green, 2000; Benyand Dolivo, 1976, Branchini et al 1980; Filippova et al., 1989).

Beetle luciferases, particularly firefly luciferase from the NorthAmerican firefly Photinus pyralis, are well known in the art. The P.pyralis luciferase (LucPpy) consists of approximately 550 amino acids of61 kDa as calculated by the protein encoded by the nucleotide sequenceof the gene. Another example of a firefly luciferase is Photurispennsylvanica firefly luciferase (LucPpe2; 545 amino acid residues,GenBank 2190534, (Ye et al., 1997)). Mutant luciferases derived fromLucPpe2 (e.g., LucPpe2 m78 (also known as 78-OB1O), SEQ ID NO: 1;LucPpe2 m90 (also known as 90-1B5), SEQ ID NO: 2, LucPpe2 m133 (alsoknown as 133-1B2), SEQ ID NO: 3, LucPpe2 m146 (also known as 146-1H2),SEQ ID NO: 4 can be used in methods of the invention. In addition, anyluciferase that meets the limitations set forth herein may be used inthe composition, method and kits of the invention. The method of makingLucPpe2 m78, LucPpe2 m90, LucPpe2 m133, and LucPpe2 m146 is disclosed inPCT/US99/30925.

In certain embodiments, isolated and/or purified luciferases can be usedin the present invention. Contaminant components of its naturalenvironment are materials that would typically interfere with diagnosticor therapeutic uses for the luciferase, and may include enzymes,hormones, and other proteinaceous or non-proteinaceous materials. Onetechnique to ascertain purity is applying SDS-PAGE analysis undernon-reducing or reducing conditions using Coomassie blue or silverstain. Isolated luciferase includes luciferase in situ withinrecombinant cells, since at least one component of the luciferasenatural environment will not be present. Luciferases can be isolatedfrom biological specimens that produce luciferase or from a cell thatexpresses an exogenous polynucleotide encoding a desired luciferase(e.g., a nucleotide encoding 78-OB10, 90-1B5, 133-1B2, or 146-1H2 (SEQID NOs 5-8, respectively)). Such techniques are well known to those ofskill in the art.

The naturally-occurring substrate for beetle luciferases is fireflyluciferin, a polyheterocyclic organic acid,D-(−)-2-(6′-hydroxy-2′-benzothiazolyl)-Δ²-thiazolin-4-carboxylic acid(luciferin). Luciferin may be isolated from nature (e.g. from fireflies)or synthesized. Synthetic luciferin can have the same structure as thenaturally occurring luciferin or can be derivatized, so long as itfunctions analogously (Bowie et al., 1973, Branchini, 2000; Craig etal., 1991; Miska and Geiger, 1987; Yang and Thomnason, 1993). Examplesof derivatives of luciferin include D-luciferin methyl ester,D-luciferyl-L-phenylalanine, D-luciferyl-L-N α-arginine, D-luciferin-Osulphate and D-luciferin-O-phosphate (Miska and Geiger, 1987), esters ofluciferases that are hydrolyzed or acted upon by esterases to luciferinby components in a sample (Craig et al., 1991; Yang and Thomason, 1993).Other examples of useful luciferin analogs include naphthyl- andquinolylluciferin, which emit light in the green and red light spectrarespectively (Branchini et al., 1989). There are multiple commercialsources for luciferin (e.g., Promega Corp. Madison, Wis., MolecularProbes, Eugene, Oreg.).

The beetle luciferase-catalyzed reaction that yields luminescence (theluciferase-luciferin reaction) involves firefly luciferin, adenosinetriphosphate (ATP), magnesium, and molecular oxygen. In the initialreaction, the firefly luciferin and ATP react to form luciferyladenylate with the elimination of inorganic pyrophosphate. The luciferyladenylate remains tightly bound to the catalytic site of luciferase.When this form of the enzyme is exposed to molecular oxygen, theenzyme-bound luciferyl adenylate is oxidized to yield oxyluciferin in anelectronically excited state. The excited oxidized luciferin emits lighton returning to the ground state.

It is contemplated that the ATP function of the reaction can beperformed by an ATP analogue (e.g., dATP). It is also contemplated thatother ions can serve as substitutes for magnesium ions (e.g., Mn2+ or Ca2+) Additionally, oxygen is a reactant of the reaction. Therefore, thereaction should not be conducted under anaerobic conditions. However, itis not generally necessary in practicing the invention to provide oxygenover and above that present in the air. Reactions can take place inclosed vessels, provided there is sufficient oxygen in the reactionsolution.

Most luciferase-luciferin reactions generate a flash of light that isshort lived. However, some of the luciferases that can be used inmethods of the invention, e.g., LucPpe2 m146 and LucPpe2 m90luciferases, under the conditions of the invention generate a“glow-type” luminescent signal with less than 50% loss of luminescenceper hour after the reagent composition is combined with the sample.

Any luciferase, luciferase variant, luciferase fragment, or variantluciferase fragment that retains the ability to generate luminescencewhen used in the reagent composition of the present invention and doesnot prevent the reagent composition from meeting the stabilityrequirements of the present invention, can be used in the presentinvention.

A full length luciferase variant will have at least about 80% amino acidsequence identity, preferably at least about 81% amino acid sequenceidentity, more preferably at least about 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acidsequence identity and most preferably at least about 99% amino acidsequence identity with a full-length native sequence luciferase sequenceand retain the ability to generate luminescence. Ordinarily, variantluciferase fragments are at least about 50 amino acids in length, oftenat least about 60 amino acids in length, more often at least about 70,80. 90, 100, 150, 200, 300, 400, 500 or 550 amino acids in length, ormore and retain the ability to generate luminescence. A luciferase,luciferase fragment, luciferase variant or variant luciferase fragmentmay be fused to other non-luciferase amino acid sequences and still befunctional in the invention.

Full length luciferase, fragments of luciferase, variants of luciferase,and variant fragments of luciferase enzyme used in the compositions andmethods of the present invention may be purified from a native source orprepared by a number of techniques, including (1) chemical synthesis,(2) enzymatic (protease) digestion of luciferase, and (3) recombinantDNA methods. Chemical synthesis methods are well known in the art, asare methods that employ proteases to cleave specific sites. To producesegments of luciferase protein segments of luciferase or luciferasevariants can be made and then expressed in a host organism, such as E.coli. Methods such as endonuclease digestion or polymerase chainreaction (PCR) allow one of skill in the art to generate an unlimitedsupply of well-defined fragments. Preferably, luciferase fragments shareat least one biological activity with native luciferase, as well ascatalytic activity, although the level of activity may vary from that ofthe native luciferase.

Any type of amino acid substitution, insertion or deletion, orcombination thereof may be used to generate a variant luciferase.However, a luciferase with a conservative amino acid substitution ismore likely to retain activity. Useful conservative substitutions areshown in Table A “Preferred substitutions.” Conservative substitutionswhereby an amino acid of one class is replaced with another amino acidof the same type fall within the scope of the invention if thesubstitution does not impair luciferase activity. TABLE A Preferredsubstitutions Original residue Exemplary substitutions Preferredsubstitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gin, Asn Lys Asn (N)Gln, His, Lys, Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn AsnGlu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile(I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L) Norleucine, Ile,Val, Met, Ile Ala, Phe Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, IleLeu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser(S) Thr ThrThr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val(V) Ile, Leu, Met, Phe, Ala, Leu Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or a-helical conformation, (2)the charge or (3) hydrophobicity, or (4) the bulk of the side chain ofthe target site might modify luciferase function. Residues are dividedinto groups based on common side-chain properties as denoted in Table B.Non-conservative substitutions entail exchanging a member of one ofthese classes for another class. TABLE B Class Amino acids HydrophobicNorleucine, Met, Ala, Val, Ley, Ile neutral hydrophilic Cys, Ser, ThrAcidic Asp, Glu Basic Asn, Gln, His, Lys, Arg disrupt chain conformationGly, Pro Aromatic Trp, Tyr, Phe

Variant luciferase genes or gene fragments can be made using methodsknown in the art such as oligonucleotide-mediated (site-directed)mutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis (Carter, 1986; Zoller and Smith, 1987), cassettemutagenesis, restriction selection mutagenesis (Wells et al, 1985) orother know techniques can be performed on the cloned DNA to produce theluciferase variant DNA (Ausubel et al., 1987; Sambrook, 1989).

1. Selected Luciferases

Any luciferase, luciferase fragment, or variants thereof that emitsphotons upon oxidation of a substrate may be used in the presentinvention. Other desirable characteristics, such as thermostability,chemostability, and signal stability, are also contemplated. Inaddition, the luciferase may be fused to another amino acid sequence andstill be functional in the present invention. Such enzymes may besynthesized in vitro or isolated from other organisms.

Naturally occurring luciferases can be found, for example, in bacteria,unicellular algae, coelenterates, beetles (other than P. pennsylvanica),fishes, and other organisms. Chemically, all luciferases involveexergonic reactions of molecular oxygen with different luciferins,resulting in photon production (Hastings, 1996; Hastings and Wilson,1976; Wilson and Hastings, 1998, Wood et al., 1989).

The use of a luciferase other than that from beetles requires anappropriate luciferin molecule that upon oxidation generates achemically and electrically unstable intermediate or a detectableenzymatic product. Other substrates may be used, as well as otherATP-dependent enzymes that produce a detectable enzymatic product.Detectable products include photons, radioactively-labeled products,insoluble or soluble chromogens, or other products that can be detectedvisually or through the use of devices.

In certain embodiments, luciferases of the invention possess catalyticactivity that depends on ATP and emits photons. In other embodiments,luciferases of the invention have enhanced chemostability in thepresence of ATPase inhibitors relative to the level of the P. pyralisluciferase (LucPpy) chemostability in the same reaction conditions. Insome embodiments, luciferases used in the compositions and methods ofthe invention generate a stable signal, i.e., they yield enhancedduration of luminescence in a luciferase reaction defined as less than50% loss of luminescence per hour relative to the luminescence at thetime the luciferase reaction was initiated. In still other embodiments,luciferases of the invention can allow for multiple analyses of a sampleover time or analysis of many samples over time, one hour after theluciferase reaction is initiated, more preferably two hours and mostpreferably four hours or more. Optionally, the luciferases used in thecompositions and methods of the invention have enhanced thermostabilityproperties. An exemplified luciferase is LucPpe2 m146 (SEQ ID NO:4).Additional examples of enzymes useful in the invention include, but arenot limited to, LucPpe2 m78 (SEQ ID NO:1), LucPpe2 m90 (SEQ ID NO: 2),and LucPpe2 m133 (SEQ ID NO:3).

The luciferases LucPpe2 m78 (SEQ ID NO: 1), LucPpe2 m90 (SEQ ID NO:2),LucPpe2m 133 (SEQ ID NO:3) and LucPpe2 m146 (SEQ ID NO.4) were generatedfrom a mutant of P. pennsylvanica (T249M). The nucleic acid sequenceencoding this protein was subjected to mutagenic methods includingrecursive mutagenesis followed by screens for thermostability, signalstability, and substrate binding and is fully described by Wood and Hall(WO 9914336, 1999).

2. Chemostable Luciferases

“Chemostable luciferases” as used herein, defines luciferases thatretain activity in the presence of compounds or conditions when thosecompounds or conditions typically inhibit ATPases and disrupt thefunction of non-chemostable luciferases such as LucPpy. The aboveidentified luciferases LucPpe2 m78 (SEQ ID NO: 1), LucPpe2 m90 (SEQ IDNO:2), LucPpe2m 133 (SEQ ID NO:3) and LucPpe2 m146 (SEQ ID NO:4), werefound herein to have enhanced chemostability to ATPase inhibitors.

Thus, in certain embodiments, luciferases of the invention include thosethat maintain at least about 30% (preferably at least about 60%, 70%,80%, 90%, 95%, 99%) enzymatic activity as measured by luminescence atleast one hour (preferably at least two hours, more preferably at leastfour hours) after contact with an amount of ATPase inhibitor, preferablya detergent, e.g., cationic detergent (preferably DTAB or BDDABr),anionic detergent (preferably deoxycholate or SDS) or zwitterionicdetergent (preferably sulfobetaine 3-10) or combination thereofsufficient to collectively reduce ATPase activity endogenous to a sampleby at least about 25% (preferably at least about 30%, even morepreferably at least about 40%, 50%, 60%. 70%, 80%, 90%, 95%, 99% or anyincrement therein) relative to the sample's ATPase activity in theabsence of the ATPase inhibitor.

The chemostability of an enzyme also may be indicated by the rate ofdecline of its activity over time. For example, shortly (0 to 10minutes) after mixing the ATPase inhibitor and the luciferase, therebycreating the reagent composition, at several subsequent time points analiquot of the reagent composition is added to a sample and relativelight unit (rlu) measurements are obtained shortly thereafter. Thesemeasurements may be graphed to determine a trend of decline in enzymeactivity in the reagent composition over time.

Chemostable luciferases (e.g., Ppe2 m78, Ppe2 m90, Ppe2 m133, and Ppe2m146) also retain activity in multi-detergent solutions. Specifically,solutions containing 0.01%, preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, most preferably 0.25% CHAPS(3-([3-Cholamidopropyl]dimethylammonio)-1-propanesulfonate) with atleast 0.01%, preferably 0.05%, 0.1%, 0.2%, and most preferably 0.3% or1.0% BDDABr, taurocholic or taurolithocholic acids, or DTAB, or 0.01%,preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4%. 0.5%, 0.6%, 0.7%. 0.8%, 0.9%,most preferably 1.0% of taurocholic or taurolithocholic acids with atleast 0.01%, preferably 0.05%, 0.1%, 0.2%, and most preferably 0.3% or1.0% BDDABr, DTAB, or CHAPS. Other multi-detergent solutions in whichLucPpe2 m78, LucPpe2 m90, LucPpe2 m133 and LucPpe2 m146 retain activityinclude 0.01%, preferably 0.05%, most preferably 0.1% TRITON X-100 withat least 0.01%, preferably 0.05%, 0.1%, 0.2%, 0.5%, most preferably 1.0%BDDABr, DTAB, or CHAPS, or 0.01%, preferably 0.05%, 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, most preferably 1.0% of taurocholicor taurolithocholic acids with at least 0.01%, preferably 0.05%, 0.1%,0.2% and most preferably 0.3 or 1.0% BDDABr, DTAB, or CHAPS; or 0.05%,1.0%, 2.0%, 4.0%, preferably 2% polyethylene glycol 400 dodecyl ether(THESIT), with at least 0.05%, preferably 0.1%, 0.2% and most preferably0.3% or 1.0% BDDABr, DTAB, or CHAPS.

3. Thermostable Luciferase

In some embodiments, a thermostable luciferase that producesluminescence or other thermostable ATP-dependent enzyme that produces adetectable signal can be used in a method of the invention, especiallyin samples that are treated with heat immediately prior to ATPdetection. A thermostable polypeptide remains active at temperaturesthat inactivate or denature other proteins. The LucPpe2 m78, LucPpe2m90, LucPpe2 m133 and LucPpe2 m146 enzymes display increasedthermostability compared to luciferases found in nature or encoded frompolynucleotides isolated from nature.

D. Kits

When the invention is supplied as a kit, the different components of thecomposition may be packaged in separate containers and admixed prior touse. The different components of the invention may comprise subsets ofthese parts and may be combined in any way that either facilitates theapplication of the invention or prolongs storage life.

The following sections are intended to provide certain examples of kitcomponents. One of skill in the art will recognize that the actualcomponents provided in a kit of the invention will vary depending onwhich particular assay is to be performed.

1. Luciferase-luciferin component

All luciferases, luciferase variants, luciferase fragments and variantluciferase fragments that catalyze an ATP-dependent reaction andgenerate luminescence are contemplated for use in the invention. Someembodiments eliminate the luciferin; for example, allowing a user tosupply a luciferin of his/her choice, or the luciferin may be providedseparately. The type of luciferin provided may vary but it must be asubstrate for the type of luciferase provided.

In one embodiment, a kit supplies the luciferase as an anhydrouspreparation. Anhydrous preparations of luciferase may be lyophilized, inwhich water is removed under vacuum, freeze-dried, crystallized, or anyother method that removes water that does not inactivate luciferase.Excipients that bulk the preparation and stabilize luciferase, such asserum albumins or Prionex, may also be included. In other embodiments,luciferase may be suspended in an aqueous composition comprisingglycerol or other solvent in which the enzyme is stable. The skilledartisan can easily determine the amounts of the various constituentsthat work in the compositions and methods of the invention.

2. Tolerance Enhancing Agents

A luminescence assay provides correlation between a biological orbiochemical process and light output. Interference by a compound maydisrupt or alter correlation to some degree. A “tolerance enhancingagent” of the invention can substantially protect luciferase activity byminimizing the effect of an interfering compound in a luciferase-basedassay system by at least about 10%, preferably about 30, 50, 60, 70, 80,90, 95, 96, 97, 98, 99 and most preferably about 100%, relative to asystem without interfering compound and bring the assay closer tocorrelation. These tolerance enhancing agents include, withoutlimitation, detergents (e.g., cationic, anionic, non-ionic and/orzwitterionic) and non-detergents. Examples of non-detergent toleranceenhancing agents include, without limitation, polyethylene glycol,polyvinyl pyrrolidone, and cyclodextrins. The selection and amount usedof a particular tolerance enhancing agent suitable for a particularluciferase-based assay may be determined by a number of ways, includingtitrating various concentrations of agent in a luciferase-based assaythat includes a known luciferase inhibitor and comparing theluminescence obtained from such an assay against a second assay thatemploys no or reduced amounts of tolerance enhancing agents. Anysuitable general luciferase inhibitor may be used as a standardinhibitor agent for identifying and screening tolerance enhancementagents. The example below provides a representative procedure forscreening of several tolerance enhancement agents and use of a knowngeneral luciferase inhibitor, isoliquirtigenin, as a standard forscreening the agents.

In practicing this invention, one or more tolerance enhancing agents maybe used in a luciferase-based assay to determine the effect of one ormore compounds on enzyme or cellular activity. The amount of toleranceenhancing tolerance enhancing agents that is present in the reagent issuch that it is effective to at least substantially protect the activityof the luciferase from interference from one or more compounds in ascreening procedure. Any suitable cationic, anionic, zwitterionic, ornon-ionic detergent may be used in this invention so long as itfunctions to enhance luciferase tolerance against one or more compoundsin a screening procedure, particularly in high throughput screeningprocedure. For instance, suitable, but non-limiting, examples ofdetergents include Tergitol (nonionic); Brij 35 (nonionic); Brij 58(nonionic); Triton X-100 (nonionic); Triton X-305 (nonionic); TritonN101(nonionic); Chaps (zwitterionic); Chapso (zwitterionic); Bigchap(nonionic); Thesit (nonionic); Pluronic L64 (nonionic); Rhodasurf 870;Chemal LA-9; Sulfonyl 465; Deoxycholate (anionic); and CTAB (cationic);Pierce CO8; Pierce C10; and Tergitol®, e.g., Tergitol NP-9®. Forluciferase-based assays for determining enzyme activity in the presenceof one or more test compounds, Tergitol (for P-450 and kinase activity),Thesit (for kinase), CHAPS (for kinase activity) are preferred.

3. ATPase Inhibitor Component

In one embodiment, particularly useful when ATP concentrations of asample are to be measured, the kit comprises a component containing oneor more ATPase inhibitors within a solution optionally containing otherfunctional components, such as buffers, defoamers, enzyme stabilizers,and the like. This component may be supplied as a working solution or asa concentrate. The ATPase inhibitor component (e.g., CTAB) may bepackaged separately. The ATPase inhibitor may be any of those describedherein above. This component may further comprise agents that chelatemetal ions that may interfere with the luciferase-luciferin reaction(e.g. EDTA, EGTA), magnesium (preferably supplied as a salt, such assulfate or chloride; or other functionally equivalent cation), defoamingagents, and inhibitors of ATP generating enzyme (e.g. NaF). Buffers thatmaintain pH of the working solution, e.g. citrate or MES (which may besupplied as a salt, such as sodium or free acid or base) or any otherappropriate buffer may be used. Instructional materials may also beenclosed in the kit as well as materials that may act as standards orcontrols, depending on the purpose of the kit.

One aspect of the invention is an ATPase inhibitor, preferably adetergent that inhibits ATPases, more preferably a detergent with acharged group, e.g., cationic detergent (preferably DTAB or BDDABr),anionic detergent (preferably deoxycholate or SDS) or zwitterionicdetergent (preferably sulfobetaine3-10). Such inhibitors prevent ATPasesendogenous to the sample from processing ATP to adenosine diphosphate(ADP) and adenosine monophosphate (AMP) before the luciferase is allowedto utilize the ATP in the sample for the luciferase-luciferin reaction.ATPase inhibitors may inactivate ATPases directly or indirectly. Theymay bind to ATPases, either in the active sites, thus preventingsubstrate binding, or denature ATPases, such as by denaturingdetergents, or they may selectively sequester ATPases from theirsubstrates.

One embodiment of the present invention uses cationic detergents such asDTAB or BDDABr detergents that act as ATPase inhibitors. However, otherATPase inhibitors are contemplated, such as other cationic detergents,anionic detergents (e.g., SDS and deoxycholate) and zwitterionicdetergents (e.g., sulfobetaine 3-10).

For DTAB or BDDABr the concentration in the reagent composition ispreferably in the range of about 0.02% to about 5.0%, more preferablyabout 0.05%, still more preferably about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%. 1.3%, 1.4% and 1.5% and mostpreferably to a final concentration of about 1.0% in the reagentcomposition.

Other non-cationic detergent ATPase inhibitors are contemplated forinclusion in the reagent composition; their requirements are that they,like DTAB, preferably inhibit at least about 25%, more preferably atleast about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, more preferably at least about 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% and most preferably about 100% of endogenousATPase activity in a sample when present in a reagent compositionwherein the reagent composition is capable of maintaining at least 30%,40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, more preferably at least about 91%, 92%, 93%. 94%, 95%, 96%, 97%,98%, 99% and most preferably about 100% activity, as measured byluminescence after the reagent composition is combined with the sample,for at least one hour, more preferably at least 2 hours compared to thereagent composition's activity just after the luciferase is combinedwith the ATPase inhibitor. Potentially suitable non-cationic detergentsthat function as ATPase inhibitors include anionic detergents(preferably SDS and deoxycholate), zwitterionic detergents (preferablysulfobetaine 3-10). The concentration of a particular ATPase inhibitorwill vary depending on the inhibitor used, and to some extent, thesample being analyzed. One of skill in the art is familiar with methodsto determine the appropriate concentration of an ATPase inhibitor forinclusion in the reagent composition; for example, they may examineluciferin-luciferase derived signals over time, comparing those samplesthat have varying concentrations of a candidate ATPase inhibitor tothose samples containing no known ATPase inhibitors.

It is fully anticipated that the most preferred concentration and eventhe concentration range functional in the methods of the invention willvary for different detergents. For example, SDS concentrationsfunctional in the methods of the invention are about 0.002%. Thefunctional concentration range for a detergent used in the presentinvention may readily be determined by one of skill in the art using themethods disclosed herein.

It is contemplated that some ATPase inhibitors, at some of theconcentrations useful in the invention, may be insoluble or have lowsolubility in aqueous solutions. These compounds may first be dissolvedin an organic solution (e.g., dimethyl sulfoxide or dimethylformamide)and then diluted into the reagent composition for use in the compositionand methods of the invention.

4. Inhibitors of ATP-Generating Enzymes

In some samples, enzymes such as kinases may be active, allowing forcontinued production of ATP. Because the ATP concentration is determinedat a specific time, if such enzymatic activity is left unchecked, thenan over-estimation of the ATP concentration will be made. To countersuch ATP-generating activity where an end-point analysis is desired,inhibitors of ATP production can be used. For end-point analysis,inactivation of the kinase reaction can be advantageous because itensures a more stable luminescent signal. Although the action of aspecific inhibitor may be incompletely understood, their usefulness isnot obviated. Examples of useful compounds include NaF, which is usefulat concentrations of at least 1 mM, preferably 2 mM to 100 mM or anyincrement therein; 2 mM is most preferred. Any such inhibitor may beused, however, if it does not adversely affect luciferase so as to takeit outside the utility of the invention. One of skill in the art willknow how to determine the appropriate concentration of such aninhibitor, whether the inhibitor is novel or well-known. Otherinhibitors of ATP generating enzymes include, but are not limited to,vanadate, paranitrophenylphosphate and dichloroacetic acid (Kiechle etal., 1980).

5. Buffers

Any buffers that maintain suitable pH for the working solution and donot interfere with the luciferase-luciferin reaction are contemplated.The preferred pH range is between about pH 4.5 and about pH 9.0, morepreferably between about pH 6.0 and about pH 8.0. For P450 activitymeasurements the luciferase assay is performed at about pH 8.4. Inaddition to MES and citrate buffers, other buffers, such as phosphatebuffered saline (PBS), Tris,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfomc acid) (HEPES),piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), borate, and any otherbuffer known to those of skill in the art may be suitable. Selection ofappropriate buffers depends on pH buffering capacity and interactionwith the luciferase-luciferin reaction.

6. Defoamers

Defoaming agents are desirable to prevent foam from interfering with thedetection of bioluminescence, especially in applications that quantifyluminescence. Such agents as MAZU® (BASF) may be organic or siliconebased. Selection of defoamers depends on their ability to eliminate foamwithout interfering with the luciferase-luciferin reaction.

7. Cations

Cations may also be included when necessary, for example in the beetleluciferase-luciferin reaction, which is dependent not only on ATP, butalso on magnesium ions. To assure luciferase activity in such cases,magnesium may be exogenously supplied. In addition to magnesium sulfate,other salts of magnesium are contemplated, such as magnesium chloride,magnesium gluconate, magnesium acetate, magnesium bromide, magnesiumcarbonate, etc. In any case, the magnesium complex must dissociate tomake Mg²⁺ ions available to the luciferase and not interfere with theluciferase-luciferin reaction. One of skill in the art is aware thatother cations may be functional in place of magnesium. These includecalcium and manganese.

In some applications, endogenous magnesium should be sufficient, inwhich cases exogenous magnesium could be eliminated.

8. Stabilizing Agents

While resistant to the action of nonionic and low concentrations ofzwitterionic detergents (Simpson and Hammond, 1991), native fireflyluciferase is inactivated by cationic detergents, such as benzalkoniumchloride, benzethonium chloride, CTAB (cetyltrimethylammonium), DTAB(dodecyltrimethylammonium bromide), and methylbenzethoniumchloride(Simpson and Hammond, 1991). Where enhanced luciferase stability isdesirable, a stabilizing agent can be provided in a kit.

The stabilizing agent can be any compound that stabilizes a luciferasefrom degradation. Suitable stabilizing agents include proteins (such asbovine serum albumin or gelatin) or detergents (preferably non-ionicdetergents, most preferably THESIT).

9. Other Agents

Other agents that may be included in a kit include agents that are knownto enhance the duration of luminescence resulting from a luciferasereaction, such as Co-enzyme A (Co A), thiol reagents, such asdithiothreitol and β mercaptoethanol (Wood, U.S. Pat. No. 5,283,179,1994; Wood, U.S. Pat. No. 5,650,289, 1997), metal ion chelators such asEDTA to prolong the signal and protease inhibitors (Schemrer, U.S. Pat.No. 5,618,682, 1997; Scheirer, U.S. Pat. No. 5,866,348, 1999) or highconcentrations of salts (Van Lune and Trer Wiel, WO 00/18953, 2000).

10. Other Kit Contents

Kits may also include reagents in separate containers that facilitatethe execution of a specific test, such as cell viability, cytotoxicity,cell proliferation, or determination of ATP concentration. For example,ATP may be supplied so that standard curves may be determined or to beused as internal controls. Also, a luciferin may be supplied so that aluciferin standard curve may be determined. The kit may supply asample-gathering component such as a membrane, filter or swab.

11. Containers or Vessels

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved, andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized luciferase orbuffer that have been packaged under a neutral, non-reacting gas, suchas nitrogen. Ampules may consist of any suitable material, such asglass, organic polymers, such as polycarbonate, polystyrene, etc.,ceramic, metal or any other material typically employed to holdreagents. Other examples of suitable containers include simple bottlesthat may be fabricated from similar tolerance enhancing agents asampules, and envelopes, that may consist of foil-lined interiors, suchas aluminum or an alloy. Plastic containers with screw tops or glasscontainers with rubber stoppers may also be used. Other containersinclude test tubes, vials, flasks, bottles, syringes, or the like.Containers may have a sterile access port, such as a bottle having astopper that can be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to mix.Removable membranes may be glass, plastic, rubber, etc.

12. Instructional Materials

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audio tape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan Internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail. In a preferred embodiment, theinstructions instruct the user to combine the luciferase with thetolerance enhancer before adding the reagent composition to a sample orto combine the tolerance enhancer with the non-luminogenic reactionbefore adding the luciferase to this reaction. E. Reagent CompositionActivity

To measure luminescence and thereby determine the reagent compositionactivity, the relative light unit (RLU) value generated by theluciferase reaction at a timepoint of interest after the reagentcomposition is combined with a sample may be measured. For example, anRLU value may be obtained by measuring the resulting luminescence from asample with a known concentration of ATP (in the case where kinaseactivity is being measured) or substrate (in the case where protease orP-450 activity is being measured) combined with the reagent compositionjust after (0-10 min) the component comprising the tolerance enhancer isadded to the component comprising the luciferase thereby creating thereagent composition. This is considered 100% activity (uninhibited)under those conditions. If, after combining a compound known to inhibitluciferase (for example, isoliquirtigenin) with the luciferase reaction,the luminescence decreases in intensity, then the relative protectionbestowed by a tolerance enhancing agent is equivalent to the relativeproximity of the intensity to the uninhibited luminescence. For example,if isoliquirtigenin inhibits luminescence in a reaction by 200-fold inthe absence of a tolerance enhancing agent, but only inhibits theluminescence by 100-fold in the presence of a compound, then thatcompound is a tolerance enhancing agent and it exerts a relativeprotection of 50%.

In practicing this invention, a reagent composition is generallycombined with a sample for the purpose of detecting a product such asATP or the occurrence of a biospecific event such as enzyme inhibitionor activation. Typically, the reagent composition comprises a luciferaseand a tolerance enhancement agent to protect luciferase activity frominterference by one or more compounds that may inhibit or interact withthe luciferase. Ambient oxygen is typically sufficient. Optionalingredients may be included in the reagent composition, depending on thetype of assay being conducted. For assays for detecting and/orquantitating ATP detection, the reagent composition includes anATP-dependent luciferase, a tolerance enhancement agent, luciferin (insaturating or sub-saturating concentrations), and a divalent cation(preferably Mg+2). Buffer and other suitable ancillary components suchas ATPase and/or enzyme stabilizers may be present as well.

For assays based on luciferin detection, ATP-dependent orATP-independent luciferases are generally used. Thus, in one embodimentof the invention involving a one step assay, the reagent compositionincludes an ATP-dependent luciferase, a luciferase pro-substrate, atolerance enhancement agent, ATP (saturating or sub-saturatingconcentration), and a divalent cation (preferably Mg+2). In analternative embodiment involving a two step assay, the reagent includesATP-dependent luciferase, ATP, and divalent cation. The luciferasepro-substrate is kept separate from the reagent and added to the sampleprior to addition of the reagent composition. In another embodiment, thereagent composition includes an ATP-independent luciferase and atolerance enhancement agent, and a luciferase pro-substrate such ascoelenterazine or coelenterazine derivative. ATP is not needed withATP-independent luciferases. In an alternative embodiment involving atwo step assay, the reagent includes ATP-independent luciferase, ATP,and divalent cation. The luciferase pro-substrate is kept separate fromthe reagent and added to the sample prior to addition of the reagentcomposition. Buffer and other suitable ancillary components such asATPase inhibitor and/or enzyme stabilizers may be present as well.

F. Detecting and Quantifying the Products of the Luciferase Reaction

A luciferase reaction results in the generation of light(“luminescence”). Users may simply visually inspect sample reactions toascertain the production of light. However, more sensitiveinstrumentations allow not only detection of faint signals, but alsoquantification of the light signal. Also contemplated are reactions inwhich non-light products are measured, according to the nature of theproducts. Any assay that results in a luminescent signal may benefitfrom the present invention. Appropriate instruments and methods for suchproducts will be apparent to the skilled artisan.

In all cases in which light is detected, specialized instruments, suchas luminometers, can read the light product of a luciferase-luciferinreaction. Any instrument that can detect light of the wavelengthsemitted by the luciferase reaction may be used. Such instruments mayread samples singularly, or in high-throughput screens, may read manysamples housed in the wells of a microwell plates (6, 24, 48, 96, 384,1536, and so on, well formats). Clearly, the devices used to measure theemitted light do not limit the invention. Other devices that can be usedinclude scintillation counters (Nguyen et al., 1988) or instrumentsinvented or adapted to be sensitive to luminescence, such as photometers(Picciolo, et al, 1977). Photographic film or X-ray film may also beused to detect luminescence. In addition, a user may visually inspect asample to qualitatively evaluate luminescence.

G. Uses for Luciferase Reactions

The invention is drawn to methods, compositions and kits that are usedto effectively and accurately detect and quantify ATP or luciferinlevels The invention comprises the addition of a single composition(reagent composition) that comprises a luciferase, and a toleranceenhancing agent and then detecting luminescence. The addition of atolerance enhancing agent of the invention can minimize the effect onluciferase of a compound, said compound being tested against a targetactivity or component (e.g., CYP450, protease, kinase, ATP level) andthereby bring luminescence into closer correlation with the targetactivity or component level.

1. Detecting Products

The methods, compositions and kits of the invention provide for thesimple qualitative or quantitative detection of ATP (or ATP analoguewhich can function as a luciferase substrate) and enzyme activity,(e.g., kinase, protease, or cytochrome P-450 activity) in a sample andfurther in the presence of one or more test compounds. In preferredembodiments, a simple qualitative experiment in which luminescence isgenerated in a sample using the invention, a tolerance enhancing agentwill minimize the effect of an interfering compound and bring theluminescence into closer correlation with the biological or biochemicalprocess being examined. Luminescence is generated using a reagentcomposition comprising luciferase such as LucPpe2 m78, LucPpe2 m90,LucPpe2 m133 or LucPpe2 m146, and possibly one or more ATPaseinhibitors. In addition, the reagent composition may further compriseone or more of the following components: luciferin, which may bereconstituted from a lyophilized preparation, (alternatively, anappropriate luciferin-analogue substrate), ATPase inhibitor(s),inhibitor(s) of ATP generating enzymes such as kinases for end-pointdeterminations, divalent cation (e.g. magnesium), enzyme stabilizingagent, and buffer.

A sample may be anything that is suspected of containing a bioactivity,e.g., kinase, protease or P450 enzyme activity, that may be susceptibleto one or more compounds in a high throughput screening procedure.Samples include solutions including one or more enzymes, cell lysates,beverages, swabs wiped on surfaces such as those of animals, plants, orinanimate objects, and the like. Other examples of samples includecompositions of a known ATP concentration or lysates of cells from anyorganism, prokaryotic or eukaryotic. Examples of prokaryotic cellsinclude E. coli, P. aeruginosa, B. subtilis, and S. typhimurium.Eukaryotic cells may be from plants, animals, fungi, insects, etc., orcultured cells from such organisms. Examples include A. thaliana andBrassica sp., Chlamydomonas sp. and Volvox sp. (plants), H. sapiens andMus sp. (animals), Saccharoymyces sp. (esp. cerevisae and pombe) andNeurospora sp. (fungi), D. melanogaster and C. elegans (insects), invitro cultured callus cells from any plant, primary cells cultured invitro from any organism (such as organ explants from, for example,rodents), mammalian cell lines such as Madin-Darby canine kidney (MDCK)and Chinese hamster ovary (CHO) cells, and insect cell lines such as sf9cells. These examples are furnished only as examples and are not meantto be limiting.

A cell lysate comprises cellular components that are no longer organizedinto a recognizable intact cellular architecture. Cell lysates may havesoluble and insoluble components, either of which may be removed beforeusing the lysate. Lysates may be prepared by any means, includingphysical disruption using sonication, a dounce, mortar and pestle,freeze-thaw cycling, or any other device or process that destroys thephysical integrity of cells; or lysis by detergents such as zwitterionicand nonionic detergents, or cationic detergents, e.g., DTAB or CTAB.Preferably, the cell lysate is produced in such a way that the integrityof the ATP concentration is preserved at the time the cells areharvested. To accurately detect ATP in a sample, enzymes that woulddegrade cellular ATP or those that would generate ATP are preferablyinhibited. In the absence of such inhibitors, there is a high risk of aninaccurate determination of ATP concentration. Inhibitors such as DTABinactivate ATPases, while other molecules such as NaF inactivateATP-generating enzyme activity. It is hypothesized, yet not fullyunderstood, that for those cell types in which NaF is effective (e g.lymphoid cells), NaF is potentially acting to inhibit (a) kinase(s).

Solutions with one or more enzymes include, without limitation, purifiedenzymes, unpurified enzymes, semi-purified enzymes, solubilized enzymes,partially solubilized enzymes, or membrane-bound enzymes.

Inhibitors of ATP-generating enzymes, those enzymes that have as aproduct or by-product ATP, such as the activity of kinases, may beincorporated into the reagent composition or kept in a separatecontainer for subsequent incorporation into the reagent composition usein cases where end-point determination of kinase activity is desired. Anexample of an effective inhibitor is NaF (Bostick, et al., 1982). Suchcompositions comprise NaF at concentrations of at least 0.5 mM,preferably at least 1 mM, more preferably at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM or any increment therein, 2mM is most preferred. Other inhibitors of ATP-generating enzymes includeother kinase inhibitors, such as vanadate, AMP, DAPP (Bostick, et al.,1982) and dichloroacetic acid (Kiechle et al., 1980).

2. Detecting and Quantifying Kinase Activity

The compositions, methods and kits of the invention permit a user todetect and quantify kinase activity by detecting and quantifying theamount of ATP consumed by the kinase in a sample by quantifying theamount of luminescence. The invention is applied to a sample ofinterest, and also to samples containing known amounts of ATP(controls). The signal generated from applying the invention to a sampleof unknown ATP concentration is correlated to signals generated eitherby internal controls (the addition of a known amount of ATP to a sampleand measuring the subsequent luminescence) or external standard curves,generated by measuring the luminescence of several samples of known ATPconcentrations and plotting them graphically. Such methods are known toskilled artisans. (Moyer and Henderson, 1983; Ronner et al., 1999;Stanley, 1989; Wood et al., 1989).

3. Effects of Compounds

The compositions, methods and kits of the present invention can beapplied to measure the effects of compounds, such as inorganics, smallmolecules, peptides, proteins, polypeptides, carbohydrates, lipids,steroids, pollutants, carcinogens, or drugs on a biospecific event whencontacted with a sample (Aiginger et al., 1980; Andreotti et al., 1995;Bradbury et al., 2000; Cree and Andreotti, 1997; Crouch et al., 1993;Kangas et al., 1984). These compounds may be catalogued in compoundlibraries, or tested singly. Such applications of the invention applycontrols in which samples are contacted with control compounds whoseeffects on ATP metabolism or enzyme function are known. Also preferably,controls include samples in which luciferase and the compound arepresent together to assure that the compound itself is not directlyaffecting luciferase activity.

The following examples are intended to illustrate the present inventionwithout limitation.

EXAMPLES Example 1 Tolerance Enhancing Effect of Detergents on theInhibition of Luciferase

In this Example, an assay that employs a luciferase reporter was used toevaluate whether known P450 enzyme inhibitors are also inhibitors ofluciferase and whether tolerance enhancing effects of several detergentscan be used to shield the luciferase against such potential inhibitorsof luciferase. There is some concern that compounds under study forcellular modulation or enzyme modulation would also affect theluciferase reaction in a luciferase based cell or enzyme reporter assaysystem. If this were true, then one would expect to obtain an increasednumber of false “hits” for an assay screen for inhibitors, for instance.Two potential luciferase inhibitors, Emodin and tyrphostin AG494, wereidentified from the “Library of Pharmaceutically Active Compounds”(LOPAC) screen. These two compounds are potential inhibitors of certaincytochrome P450 enzymes. The objective of the current experiment was todetermine if Emodin and Tyrphostin AG494 are also inhibitors of theluciferase reaction, and further to determine whether the presence ofdetergents could alleviate any inhibition on luciferase caused by thesecompounds. Luciferase activity was assayed in the presence of luciferin,with or without the compounds, and in the absence or presence of fivedifferent detergents at two different concentrations.

First, three luciferin-inhibitor 2× mixes were prepared. Each contains100 mM KPO₄, 20 nM luciferin, 0.1 mg/ml Sf9 control cell microsomalmembranes (Available from BD Gentest, Bedford, Mass.); these membraneswere used to mimic assay conditions of particular interest, a P450assay, without the presence of the P-450 enzyme as P450s are commonlyexpressed recombinantly in sf9 cell microsomal membranes), ±10 uM Emodinor 10 uM Tyrphostin AG494. The control mix containing no inhibitor wasalso prepared.

Second, eleven 2× luciferin detection reagents were reconstituted from alyophilized cake containing UltraGlo® luciferase (100 micrograms/ml,available from Promega Corp.), ATP (400 micromolar), and excipient (0.4%Prionex® (Pentapharm, Basel, Switzerland)) using a buffer (200 mMtricine, pH 8.4, 20 mM MgSO₄). The final 2× reaction mixtures containedone of the following:

-   1/ No detergent-   2/ 0.2% TOMAH-   3/ 2% TOMAH-   4/ 0.2% Tergitol NP9-   5/ 2% Tergitol NP9-   6/ 0.2% Thesit-   7/ 2% Thesit-   8/ 0.2% CHAPS-   9/ 2% CHAPS-   10/ 0.2% Triton X-100-   11/2% Triton X-100    The final concentration will be either 0%, 0.1% or 1% detergent.    Finally, 50 microliters of each luciferin-inhibitor mix was combined    with 50 microliters of each luciferin detection reagent in    triplicate on white luminometer 96-well plates, mixed, and read on a    BMG Fluostar luminometer (BMG). The results are expressed in both    RLU and % control and are illustrated in FIG. 1.

FIG. 1 illustrates the relief of inhibition on luciferase by detergentsin a standard luciferase-based reaction in the presence of luciferaseinhibitors tryphostin or emodin. Part (a) compares RLU of control (nodetergent) against reaction mixtures that contain 0.1 or 1% of Tomah,Tergitol (NP9), Thesit, CHAPS, or Triton X-100 as a function of rluvalues. Part (b) is an analysis that compares the relative % of controlagainst reaction mixtures that contain 0.1 or 1% of Tomah, Tergitol,Thesit, CHAPS, or Triton X-100.

Example 2 Minimization of False Hit Occurrence Using ToleranceEnhancement Agents

In this Example, screening of a commercially available library, LOPAC™library (Library of Pharmaceutically Active Compounds, available fromSigma Corp., St. Louis, Mo., USA) was performed to determine the effectof tolerance enhancing agents in protecting the luciferase reaction in acell based or cell-free luciferase-based assay against potentialinhibition by the drug compounds. The LOPAC library contains 640compounds whose pharmaceutical activities are known. This library iscommonly used to validate screening protocols that are underdevelopment.

From a LOPAC screen of 640 compounds, 189 compounds were identified asinhibiting luminescence from all of three cytochrome P450 isoforms(CYP1A2, CYP1A1, CYP2C9) by >10% of an untreated control. It wasreasoned that these might be luciferase inhibitors. If any one of thethree CYP450 reactions were not apparently inhibited, then inhibition ofthe luciferase detection system could be ruled out. For the 189compounds, there were three possible interpretations of the result. One,the compounds are true inhibitors of the P450s; two, the compounds areinhibiting the luciferase, meaning they are false hits; three, thecompounds are inhibiting CYP450 and luciferase. To distinguish betweenthese possibilities, the compounds were screened in the presence ofluciferin, without any P450 enzyme. If a compound continues to score asa hit, then it is inhibiting luciferase.

First, in duplicate wells on 96-well white luminometer plates, 2×luciferin-compound mixtures were prepared in 50 microliter, containing10 uM of each compound, 40 nM luciferin, and 100 mM KPO₄ as described inExample 1. Each plate also contained control wells in duplicate, whereno compound was added (vehicle alone).

Second, 2× luciferin detection reagents were prepared for each reaction,in triplicate as described in Example 1. The 2× reagents further containof the following:

-   1/No detergent-   2/ 0.2% Tergitol NP9-   3/ 2% Tergitol NP9    The final detergent concentration will be 0.1% or 1% detergent.

Finally, 50 microliters of each luciferin-inhibitor mix is combined with50 microliters of each luciferin detection reagent in duplicate on whiteluminometer 96-well plates, mixed, let sit at room temperature for 15minutes, and then read on a BMG Fluostar luminometer.

A compound would be considered a “hit” if it is more or less thanaverage ±3× standard deviation (SD) of the control (99% confidenceinterval). On the accompanying graphs (FIGS. 2(a) to 2(d)) each diamondrepresents one compound, the middle line represents the average of thecontrol (containing no inhibitor), and the two lines outside the middleline represent average ±3×SD of the control.

In the absence of any detergent, five compounds were identified asinhibitors of luciferase. Thus in a screen that also contains an enzymeof interest (such as cytochrome P450), these agent would be identifiedas “false” hits. However in the presence of 0.1% Tergitol, there areonly four false hits. Finally, in the presence of 1% Tergitol, only onehit remains, while the other four either no longer inhibit or barelyinhibit luciferase.

The results shown in FIGS. 2(A)-(D) demonstrate that it is advantageousto screen drug libraries in the presence of a tolerance enhancing agentsuch as a detergent because it helps minimize the number of false hits.

Example 3 Tolerance Enhancing Effect of Detergents and Non-Detergents onthe Inhibition of Firefly and Renilla Luciferases

In this experiment, several tolerance enhancing agents were evaluatedfor their ability to protect luciferases from a known luciferaseinhibitor, isoliquirtigenin (ILT) (100 μM, Sigma Chemical). ILT wasadded to luciferase reactions and the potential tolerance enhancingagents were evaluated for their ability to protect the luciferase frominhibition.

The basic firefly luciferase reactions contained 20 mM HEPES (SigmaChemical) at pH 8.0, 6.4 mM Magnesium Sulfate (Fisher Scientific), 20 mMDithiothreitol (City Chemical), 0.63 mM Coenzyme A (PharmaciaBiochemical), 0.4 mM EDTA (Sigma Chemical), 4.0 μM Luciferin (PromegaBiosciences), 0.56 mM ATP (Pharmacia Biochemical), and 1.3×10-6 mg/mlfirefly luciferase (QuantiLum®, Promega Corporation).

The basic Renilla luciferase reactions contained 100 mM PotassiumPhosphate buffer (Sigma Chemical) at pH 7.4, 500 mM Sodium Chloride(Fisher Scientific), 1.0 mM EDTA (Sigma Chemical), 200 nM Coelenterazine(Promega Biosciences), 0.1% Gelatin (Sigma Chemical) and 0.12 nmol/mlRenilla luciferase (ChemiCon Chemicals).

ILT was solubilized at 100 mM in dimethyl sulfoxide (Sigma Chemical).ILT was diluted 1:1000 and the protectors were diluted into the reactionmix before the reaction was initiated by the addition of luciferase.

Reaction volume for each sample was 100 μl. Luminescence was integratedfor 0.5 seconds per sample in a Turner Biosystems Veritas luminometerimmediately after luciferase was added to the reactions and thereactions were mixed. All measurements were done in triplicate.

The relative protection provided by each compound is calculated asdifference between the luminescence generated in the presence and theabsence of the protector divided by the total amount of inhibition asrepresented by the following equation:(L1−L2)/(L3−L4)wherein L1 represents luminescence with inhibitor and toleranceenhancing agent; L2 represents luminescence with inhibitor and notolerance enhancing agent; L3 represents luminescence with no inhibitorand no tolerance enhancing agent; and L4 represents luminescence withinhibitor and no tolerance enhancing agent.

The relative protection in the presence of no tolerance enhancing agent,therefore, is 0% and in the absence of an inhibitor is 100%. Thereactions used to generate luminescence in the absence of eitherinhibitor or tolerance enhancing agent still contained the solventutilized to solubilize the inhibitor with which the reaction was to becompared.

Firefly Luciferase Relative Reaction Protection Protector SourceConcentration Against ILT Firefly luciferase α-Cyclodextrin SigmaChemical   7 Mm 61% (Cyclohexaamylose) Sucrose Sigma Chemical  0.5%(w:v) 32% Polyvinyl Sigma Chemical  0.1% w:v 69% pyrrolidone CHAPS SigmaChemical 0.02% 18% CHAPS Sigma Chemical 0.10% 57% Deoxycholate SigmaChemical 0.02% 81% Tergitol NP-9 Sigma Chemical 0.02% 88% CTAB SigmaChemical 0.02% 100%  Renilla luciferase α-Cyclodextrin Sigma Chemical 1.4 mM 60% (Cyclohexaamylose) Sucrose Sigma Chemical 0.10% (w:v) 24%Polyvinyl Sigma Chemical 0.10% w:v 74% pyrrolidone CHAPS Sigma Chemical0.10% 14% CHAPS Sigma Chemical 0.50% 97% Deoxycholate Sigma Chemical0.10% 72% Tergitol NP-9 Sigma Chemical 0.10% 97% CTAB Sigma Chemical0.10% 95%As seen in the data above, compounds that convey protection to theluciferase from the inhibitors can be easily determined. Also, thetolerance enhancing agents are not equally effective and theconcentration of the tolerance enhancing agent affects the level ofprotection achieved.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions and omissions, that maybe made in what has been disclosed herein without departing from thespirit of the invention. Accordingly, it is intended that thesemodifications also be encompassed by the present invention and that thescope of the present invention be limited solely by the broadestinterpretation that lawfully can be accorded the appended claims.

1. A method for enhancing the tolerance of an assay reagent to compoundsin an assay sample not containing living cells, said assay reagentcomprising a luciferase enzyme, and said method comprising contactingsaid luciferase enzyme with a tolerance enhancement agent in an amountsufficient to substantially protect luciferase enzymatic activity frominterference of the compound.
 2. The method according to claim 1 whereinthe tolerance enhancement agent comprises a detergent or anon-detergent.
 3. The method according to claim 2 where the toleranceenhancement agent comprises a cationic, anionic, non-ionic, orzwitterionic detergent.
 4. The method according to claim 2 wherein thedetergent comprises Tergitol®, Brij 35®, Brij 58®, Triton X-100®, TritonX-305®, Triton N101®, Chaps®, Chapso®, Bigchap®, Thesit®, Pluronic L64®,Rhodasurf 870®, Chemal LA-9®, Sulfonyl 465®, deoxycholate, CTAB, PierceC08®, or Pierce C10 detergent.
 5. The method according to claim 4wherein the detergent is Tergitol® NP-9.
 6. The method according toclaim 2 wherein the non-detergent comprises polyethylene glycol,polyvinyl pyridine, crown ether, and cyclodextrin.
 7. A method ofdetermining the effect of a compound on a non-luminogenic enzymeactivity in an assay sample not containing living cells, comprising: (a)providing a compound and a luminogenic molecule, wherein the luminogenicmolecule is a substrate of the non-luminogenic enzyme and apro-substrate of a luciferase enzyme; (b) contacting the compound andthe non-luminogenic enzyme so as to produce a first reaction mixture;(c) contacting the first reaction mixture with a reagent compositioncomprising luciferase, the luminogenic molecule, and a toleranceenhancement agent so as to produce a second reaction mixture, whereinsaid tolerance enhancement agent being present in an amount effective tosubstantially protect the activity of the luciferase from interferencefrom the compound; (d) detecting luminescence in the second reactionmixture; and (e) determining the effect of the compound, if any, on thenon-luminescent enzyme activity by comparing the detected luminescenceto the luminescence of a similar reaction mixture not containing thecompound, or containing the compound at a different concentration.
 8. Amethod of determining the effect of a compound on a non-luminogenicenzyme activity in an assay sample not containing living cells,comprising: (a) providing a compound and a luminogenic molecule, whereinthe luminogenic molecule is a substrate of the non-luminogenic enzymeand a pro-substrate of a luciferase enzyme; (b) contacting the compound,and the non-luminogenic enzyme so as to produce a first reactionmixture; (c) contacting the first reaction mixture with the luminogenicmolecule so as to produce a second reaction mixture; (d) contacting thesecond reaction mixture with a reagent composition comprising luciferaseand a tolerance enhancement agent so as to produce a third reactionmixture, wherein said tolerance enhancement agent being present in anamount effective to substantially protect the activity of the luciferasefrom interference from the compound; (e) detecting luminescence in thethird reaction mixture; and (f) determining the effect of the compound,if any, on the non-luminescent enzyme activity by comparing the detectedluminescence to the luminescence of a similar reaction mixture notcontaining the compound, or containing the compound at a differentconcentration.
 9. A method of determining the effect of a compound on anon-luminogenic enzyme activity in an assay sample not containing livingcells, comprising: (a) providing a luminogenic molecule and a compoundfor testing, the luminogenic molecule is a substrate for thenon-luminogenic enzyme and a pro-substrate for luciferase; (b)contacting the compound, the luminogenic molecule and a non-luminogenicenzyme so as to produce a first reaction mixture; (c) contacting thefirst reaction mixture with a reagent composition comprising luciferase,and a tolerance enhancement agent so as to produce a second reactionmixture, wherein said tolerance enhancement agent being present in anamount effective to at least substantially protect the activity of theluciferase from interference from the compound; (d) detectingluminescence in the second reaction mixture; and (e) determining theeffect of the compound, if any, on the non-luminescent enzyme activityby comparing the detected luminescence to the luminescence of a similarreaction mixture not containing the compound, or containing the compoundat a different concentration.
 10. A method of determining the effect ofa compound on a non-luminogenic enzyme activity in an assay sample notcontaining living cells, comprising: (a) providing a compound fortesting, a substrate for the non-luminogenic enzyme, a non-luminogenicenzyme, and ATP or ADP; (b) contacting the compound, the substrate, ATPor ADP, and a non-luminogenic enzyme so as to produce a first reactionmixture; (c) contacting the first reaction mixture with a reagentcomposition comprising luciferase, luciferin, and a toleranceenhancement agent so as to produce a second reaction mixture, whereinsaid tolerance enhancement agent being present in an amount effective tosubstantially protect the activity of the luciferase from interferencefrom the compound; (d) detecting luminescence in the second reactionmixture; and (e) determining the effect of the compound, if any, on thenon-luminescent enzyme activity by comparing the detected luminescenceto the luminescence of a similar reaction mixture not containing thecompound, or containing the compound at a different concentration.
 11. Amethod of determining the effect of a compound on ATP generating enzymeactivity in a sample not containing living cells, comprising: (a)providing a ADP and a compound for testing; (b) contacting the compound,ADP and a sample so as to produce a first reaction mixture; (c)contacting the first reaction mixture with a reagent compositioncomprising luciferase, luciferin, and a tolerance enhancing agent so asto produce a second reaction mixture, wherein said tolerance enhancementagent being present in an amount effective to substantially protect theactivity of the luciferase from interference from the compound; (d)detecting luminescence in the second reaction mixture; and (e)determining the effect of the compound, if any, on the non-luminescentenzyme activity by comparing the detected luminescence to theluminescence of a similar reaction mixture not containing the compound,or containing the compound at a different concentration.
 12. The methodaccording to any one of claims 7 to 11 wherein the steps are conductedconsecutively.
 13. The method according to any one of claims 7 to 11wherein steps (b) and (c) are conducted simultaneously.
 14. The methodaccording to claim 8 wherein steps (b), (c), and (d) are conductedsimultaneously.
 15. The method according to claim 8 wherein steps (c)and (d) are conducted simultaneously.
 16. The method according to anyone of claims 7 to 11 wherein the tolerance enhancement agent comprisesa detergent or a non-detergent.
 17. The method according to claim 16where the tolerance enhancement agent comprises a cationic, anionic,non-ionic, or zwitterionic detergent.
 18. The method according to claim17 wherein the detergent comprises Tergitol®, Brij 35®, Brij 58®, TritonX-100®, Triton X-305®, Triton N101®, Chaps®, Chapso®, Bigchap®, Thesit®,Pluronic L64®, Rhodasurf 870®, Chemal LA-9®, Sulfonyl 465®,deoxycholate, CTAB, Pierce C08®, or Pierce C l10 detergent.
 19. Themethod according to claim 16 wherein the non-detergent comprises,polyethylene glycol, polyvinyl pyridine, crown ether, and cyclodextrin.20. The method of any one of claims 7 to 11 wherein the non-luminogenicenzyme is a protease.
 21. The method according to claim 20 wherein theprotease is trypsin, trypsinase, or caspase.
 22. The method according toclaim 21 wherein caspase comprises caspase-3, caspase 7, caspase 8, orcaspase-9.
 23. The method according to claim 20 wherein the luminogenicsubstrate comprises an aminoluciferin amino modified with an amino acidor peptide or a carboxylprotected derivative thereof.
 24. The methodaccording to any one of claims 8 or 9 wherein the non-luminogenic enzymeis a cytochrome P-450 enzyme.
 25. The method according to claim 24wherein the luminogenic substrate is a D-luciferin derivative.
 26. Themethod according to claim 24 wherein the tolerance enhancement agent isTergitol® detergent.
 27. The method according to claim 26 wherein theTergitol® detergent is Tergitol NP-9® detergent.
 28. The methodaccording to claim 10 wherein the non-luminogenic enzyme is a kinase.29. The method according to claim 28 wherein the detergent is Tergitol®,Thesit®, or Chaps® detergent.
 30. The method according to claim 10wherein the compound and enzyme are contacted for a first predeterminedtime period prior to contact with the substrate and ATP or ADP.
 31. Themethod according to claim 30 wherein the substrate and ATP or ADP areadded sequentially or simultaneously.
 32. The method according to claim11 wherein the ATP generating enzyme is a kinase or phosphatase.
 33. Themethod according to claim 32 wherein the tolerance enhancement agentcomprises Tergitol®, Thesit®, or Chaps® detergent.
 34. A method ofdetermining the effect of a compound on a kinase enzyme activity in anassay sample not containing living cells, comprising: (a) providing acompound for testing, a kinase substrate, a kinase enzyme, and ATP orADP; (b) contacting the compound, the substrate, ATP or ADP, and kinaseenzyme so as to produce a first reaction mixture; (c) contacting thefirst reaction mixture with a reagent composition comprising luciferase,luciferin, and a tolerance enhancement agent so as to produce a secondreaction mixture, wherein said tolerance enhancement agent being presentin an amount effective to substantially protect the activity of theluciferase from interference from the compound; (d) detectingluminescence in the second reaction mixture; and (e) determining theeffect of the compound, if any, on kinase enzyme activity by comparingthe detected luminescence to the luminescence of a similar reactionmixture not containing the compound, or containing the compound at adifferent concentration.
 35. The method according to claim 34 whereinthe kinase enzyme is a protein kinase.
 36. The method according to claim35 wherein the tolerance enhancement agent comprises a Tergitol®,Thesit®, or Chaps® detergent.
 37. The method according to claim 34wherein the compound and kinase enzyme are contacted for a firstpredetermined time period prior to contact with the substrate and ATP orADP.
 38. The method according to claim 34 wherein the substrate and ATPor ADP are added sequentially or simultaneously.
 39. The methodaccording to claim 34 wherein the steps are carried out sequentially.40. The method according to claim 34 wherein step(b) and step(c) arecarried out simultaneously.
 41. A kit comprising: (a) a toleranceenhancement agent for substantially protecting luciferase activity frominterference from a test compound; (b) a luciferase enzyme; (c) optionalbuffer reagents; and (d) optional directions for using the kit.
 42. Thekit according to claim 41 further comprising ATP.
 43. The kit accordingto claim 41 wherein the tolerance enhancement agent comprises adetergent or a non-detergent.
 44. The kit according to claim 43 whereinthe detergent comprises Tergitol®, Brij 35®, Brij 58®, Triton X-100®,Triton X-305®, Triton N101®, ChapsE, Chapso®, Bigchap®, Thesit®,Pluronic L64®, Rhodasurf 870®, Chemal LA-9®, Sulfonyl 465®,deoxycholate, CTAB, Pierce C08®, or Pierce C10® detergent.
 45. The kitaccording to claim 43 wherein the non-detergent comprises polyethyleneglycol, polyvinyl pyridine, crown ether, and cyclodextrin.
 46. The kitaccording to claim 41, further comprising optional luciferase substrateor prosubstrate.